H1 Binding Research Articles

Conformational flexibility of histone variant CENP-ACse4 is regulated by histone H4: A mechanism to stabilize soluble Cse4

The histone variant CENP-ACse4 is a core component of the specialized nucleosome at the centromere in budding yeast and is required for genomic integrity. Accordingly, the levels of Cse4 in cells are tightly regulated, primarily by ubiquitin-mediated proteolysis. However, structural transitions in Cse4 that regulate its centromeric localization and interaction with regulatory components are poorly understood. Using time-resolved fluorescence, NMR, and molecular dynamics simulations, we show here that soluble Cse4 can exist in a "closed" conformation, inaccessible to various regulatory components. We further determined that binding of its obligate partner, histone H4, alters the interdomain interaction within Cse4, enabling an "open" state that is susceptible to proteolysis. This dynamic model allows kinetochore formation only in the presence of H4, as the Cse4 N terminus, which is required for interaction with other centromeric components, is unavailable in the absence of H4. The specific requirement of H4 binding for the conformational regulation of Cse4 suggests a structure-based regulatory mechanism for Cse4 localization. Our data suggested a novel structural transition-based mechanism where conformational flexibility of the Cse4 N terminus can control Cse4 levels in the yeast cell and prevent Cse4 from interacting with kinetochore components at ectopic locations for formation of premature kinetochore assembly.

UBN1/2 of HIRA complex is responsible for recognition and deposition of H3.3 at cis-regulatory elements of genes in mouse ES cells

BackgroundH3.3 is an ancient and conserved H3 variant and plays essential roles in transcriptional regulation. HIRA complex, which is composed of HIRA, UBN1 or UBN2, and Cabin1, is a H3.3 specific chaperone complex. However, it still remains largely uncharacterized how HIRA complex specifically recognizes and deposits H3.3 to the chromatin, such as promoters and enhancers.ResultsIn this study, we demonstrate that the UBN1 or UBN2 subunit is mainly responsible for specific recognition and direct binding of H3.3 by the HIRA complex. While the HIRA subunit can enhance the binding affinity of UBN1 toward H3.3, Cabin1 subunit cannot. We also demonstrate that both Ala87 and Gly90 residues of H3.3 are required and sufficient for the specific recognition and binding by UBN1. ChIP-seq studies reveal that two independent HIRA complexes (UBN1-HIRA and UBN2-HIRA) can cooperatively deposit H3.3 to cis-regulatory regions, including active promoters and active enhancers in mouse embryonic stem (mES) cells. Importantly, disruption of histone chaperone activities of UBN1 and UBN2 by FID/AAA mutation results in the defect of H3.3 deposition at promoters of developmental genes involved in neural differentiation, and subsequently causes the failure of activation of these genes during neural differentiation of mES cells.ConclusionTogether, our results provide novel insights into the mechanism by which the HIRA complex specifically recognizes and deposits H3.3 at promoters and enhancers of developmental genes, which plays a critical role in neural differentiation of mES cells.

Dynamic placement of the linker histone H1 associated with nucleosome arrangement and gene transcription in early Drosophila embryonic development

The linker histone H1 is critical to maintenance of higher-order chromatin structures and to gene expression regulation. However, H1 dynamics and its functions in embryonic development remain unresolved. Here, we profiled gene expression, nucleosome positions, and H1 locations in early Drosophila embryos. The results show that H1 binding is positively correlated with the stability of beads-on-a-string nucleosome organization likely through stabilizing nucleosome positioning and maintaining nucleosome spacing. Strikingly, nucleosomes with H1 placement deviating to the left or the right relative to the dyad shift to the left or the right, respectively, during early Drosophila embryonic development. H1 occupancy on genic nucleosomes is inversely correlated with nucleosome distance to the transcription start sites. This inverse correlation reduces as gene transcription levels decrease. Additionally, H1 occupancy is lower at the 5′ border of genic nucleosomes than that at the 3′ border. This asymmetrical pattern of H1 occupancy on genic nucleosomes diminishes as gene transcription levels decrease. These findings shed new lights into how H1 placement dynamics correlates with nucleosome positioning and gene transcription during early Drosophila embryonic development.

Histone H1 acetylation at lysine 85 regulates chromatin condensation and genome stability upon DNA damage.

Linker histone H1 has a key role in maintaining higher order chromatin structure and genome stability, but how H1 functions in these processes is elusive. Here, we report that acetylation of lysine 85 (K85) within the H1 globular domain is a critical post-translational modification that regulates chromatin organization. H1K85 is dynamically acetylated by the acetyltransferase PCAF in response to DNA damage, and this effect is counterbalanced by the histone deacetylase HDAC1. Notably, an acetylation-mimic mutation of H1K85 (H1K85Q) alters H1 binding to the nucleosome and leads to condensed chromatin as a result of increased H1 binding to core histones. In addition, H1K85 acetylation promotes heterochromatin protein 1 (HP1) recruitment to facilitate chromatin compaction. Consequently, H1K85 mutation leads to genomic instability and decreased cell survival upon DNA damage. Together, our data suggest a novel model whereby H1K85 acetylation regulates chromatin structure and preserves chromosome integrity upon DNA damage.

Genome-wide distribution of linker histone H1.0 is independent of MeCP2.

Previous studies suggested that MeCP2 competes with linker histone H1, but this hypothesis has never been tested in vivo. Here, we performed ChIP-Seq of Flag-tagged-H1.0 in mouse forebrain excitatory neurons. Unexpectedly, Flag-H1.0 and MeCP2 occupied similar genomic regions and the Flag-H1.0 binding was not changed upon MeCP2 depletion. Furthermore, mild overexpression of H1.0 did not alter MeCP2 binding, suggesting that the functional binding of MeCP2 and H1.0 are largely independent.

Investigation into the Binding Interactions of Saccharomyces cerevisiae Histone H1 with Holliday Junction.

In eukaryotic organisms, DNA is tightly packaged into chromatin through association with proteins to form a highly condensed structure called the chromosome. Linker histone H1 binds DNA at the nucleosome entry and exit points and potentially regulates chromosome structure and gene expression. Unlike histone H1 found in many higher order eukaryotic organisms, which contain only one globular domain, Saccharomyces cerevisiae (S. cerevisiae) histone H1 contains two distinct globular domains. The importance of the two individual globular domains with respect to binding and recognition remains unclear. Four‐way DNA junctions (4WJs) are a structural model of the naturally occurring Holliday junction present during homologous recombination and repair, and H1 has previously been shown to bind 4WJs preferentially. We propose that H1 binding to 4WJs is similar structurally to how H1 binds to the nucleosome, where the arms of the junction resemble the DNA crossover strands of the nucleosome entry and exit site. We are investigating whether yeast histone H1 exhibits preferential binding to 4WJ over that of duplex DNA. We are also examining how the presence of the two globular domains affects H1 binding to both 4WJs and duplex DNA.To investigate the binding affinity of yeast H1 to DNA, S. cerevisiae H1 was purified from an E. coli strain overexpressing the protein and characterized through DNA binding assays. Although H1 has previously been shown to preferentially bind to junction over duplex DNA, preliminary results of our DNA binding studies suggest that H1 shows only a slight preference for junction DNA. Results of gel mobility shift assays (GMSA) show that H1 binds to duplex DNA with a KD of 33.6 nM, and junction with a KD of 24.5 nM. Fluorescence intensity experiments are relatively consistent with GMSA results. Specifically, we determined that H1 binds duplex with a KD of 18.8 ± 2.1 nM and junction with a KD of 7.7 ± 1.1 nM. These solution results suggest that H1 has a 2–3 fold higher affinity for the 4WJ relative to duplex DNA. Under similar solution conditions, mouse histone H1 binds to duplex and junction DNA with comparable affinity. Therefore, our current results suggest that the binding of yeast H1 to both junction and duplex is similar to mouse histone H1, despite the presence of its second globular domain.The effect of the two globular domains of S. cerevisiae H1 on DNA binding was further investigated through the creation of two mutant strains, each containing only one of the two globular domains. These mutants have been overexpressed and purified from E. Coli to examine the differential binding properties of the individual domains. The binding affinities of these constructs for junction and duplex DNA will be presented.Support or Funding InformationThis work was supported by the ASBMB Summer Undergraduate Research Award and the National Science Foundation (MCB‐0843656)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Quercetin Prevents In Vivo and In Vitro Myocardial Hypertrophy Through the Proteasome-GSK-3 Pathway.

Quercetin, a flavonoid, has been reported to ameliorate cardiovascular diseases, such as cardiac hypertrophy. However, the mechanism is not completely understood. In this study, a mechanism related to proteasome-glycogen synthesis kinase 3 (GSK-3) was elucidated in rats and primary neonatal cardiomyocytes. Rats were subjected to sham or constriction of abdominal aorta surgery groups and treated with or without quercetin for 8weeks. Angiotensin II (Ang II)-induced primary cardiomyocytes were cultured with quercetin treatment or not for 48h. Echocardiography, real-time RT-PCR, histology, immunofluorescence, and Western blotting were conducted. Proteasome activities were also detected using a fluorescent peptide substrate. Echocardiography showed that quercetin prevented constriction of abdominal aorta-induced cardiac hypertrophy and improved the cardiac diastolic function. In addition, quercetin also significantly reduced the Ang II-induced hypertrophic surface area and atrial natriuretic factor (ANF) mRNA level in primary cardiomyocytes. Proteasome activities were obviously inhibited in the quercetin-treated group both in vivo and in vitro. Quercetin also decreased the levels of proteasome subunit beta type (PSMB) 1, PSMB2, and PSMB5 of the 20S proteasome as well as the levels of proteasome regulatory particle (Rpt) 1 and Rpt4 of the 19S proteasome. In particular, the PSMB5 level in the nucleus was reduced after quercetin treatment. Furthermore, phosphorylated GSK-3α/β (inactivation of GSK-3) was decreased, which means that GSK-3 activity was increased. The phosphorylation levels of upstream AKT (PKB (protein kinase B)) and liver kinase B1/AMP activated protein kinase (LKB1/AMPKα) and those of downstream extracellular signal-regulated kinase (ERK), histone H3, β-catenin, and GATA binding protein 4 (GATA4) were reduced after quercetin treatment, while hypertrophy was reversed after treatment with the GSK-3 inhibitor. In summary, quercetin prevents cardiac hypertrophy, which is related to proteasome inhibition and activation of GSK-3α/β. Upstream (AKT, LKB1/AMPKα) and downstream hypertrophic factors, such as ERK, histone H3, β-catenin, and GATA4, may also be involved.

Using a model comparison approach to describe the assembly pathway for histone H1.

Histones H1 or linker histones are highly dynamic proteins that diffuse throughout the cell nucleus and associate with chromatin (DNA and associated proteins). This binding interaction of histone H1 with the chromatin is thought to regulate chromatin organization and DNA accessibility to transcription factors and has been proven to involve a kinetic process characterized by a population that associates weakly with chromatin and rapidly dissociates and another population that resides at a binding site for up to several minutes before dissociating. When considering differences between these two classes of interactions in a mathematical model for the purpose of describing and quantifying the dynamics of histone H1, it becomes apparent that there could be several assembly pathways that explain the kinetic data obtained in living cells. In this work, we model these different pathways using systems of reaction-diffusion equations and carry out a model comparison analysis using FRAP (fluorescence recovery after photobleaching) experimental data from different histone H1 variants to determine the most feasible mechanism to explain histone H1 binding to chromatin. The analysis favors four different chromatin assembly pathways for histone H1 which share common features and provide meaningful biological information on histone H1 dynamics. We show, using perturbation analysis, that the explicit consideration of high- and low-affinity associations of histone H1 with chromatin in the favored assembly pathways improves the interpretation of histone H1 experimental FRAP data. To illustrate the results, we use one of the favored models to assess the kinetic changes of histone H1 after core histone hyperacetylation, and conclude that this post-transcriptional modification does not affect significantly the transition of histone H1 from a weakly bound state to a tightly bound state.

Sulforaphane restores acetyl-histone H3 binding to Bcl-2 promoter and prevents apoptosis in ethanol-exposed neural crest cells and mouse embryos

Sulforaphane (SFN) is an isothiocyanate derived from cruciferous vegetables. SFN's cytoprotective properties have been demonstrated in several models associated with a variety of disorders. Our recent studies have shown that SFN protects against ethanol-induced oxidative stress and apoptosis in neural crest cells (NCCs), an ethanol-sensitive cell population implicated in Fetal Alcohol Spectrum Disorders (FASD). This study is designed to test the hypothesis that SFN can prevent ethanol-induced apoptosis in NCCs by inhibiting HDAC and increasing histone acetylation at the Bcl-2 promoter. We found that exposure to 50mM ethanol resulted in a significant increase in HDAC activities in NCCs. Treatment with SFN decreased the activities of HDAC in ethanol-exposed NCCs. We also found that SFN treatment significantly increased the expression of acetyl-histone H3 in NCCs treated with ethanol. ChIP-qPCR assay revealed that ethanol exposure significantly decreased acetyl-histone H3 binding to the Bcl-2 promoter while supplementing with SFN reversed the ethanol-induced reduction in acetyl-histone H3 binding to the Bcl-2 promoter. In addition, SFN treatment restored the expression of Bcl-2 in ethanol-exposed NCCs and diminished ethanol-induced apoptosis in NCCs. Treatment with SFN also significantly diminished apoptosis in mouse embryos exposed to ethanol in vivo. These results demonstrate that SFN can epigenetically restore the expression of Bcl-2 and attenuate ethanol-induced apoptosis by increasing histone acetylation at the Bcl-2 promoter and suggest that SFN may prevent FASD through epigenetic regulation of the expression of anti-apoptotic genes.

A histone-mimicking interdomain linker in a multidomain protein modulates multivalent histone binding

N-terminal histone tails are subject to many posttranslational modifications that are recognized by and interact with designated reader domains in histone-binding proteins. BROMO domain adjacent to zinc finger 2B (BAZ2B) is a multidomain histone-binding protein that contains two histone reader modules, a plant homeodomain (PHD) and a bromodomain (BRD), linked by a largely disordered linker. Although previous studies have reported specificity of the PHD domain for the unmodified N terminus of histone H3 and of the BRD domain for H3 acetylated at Lys14 (H3K14ac), the exact mode of H3 binding by BAZ2B and its regulation are underexplored. Here, using isothermal titration calorimetry and NMR spectroscopy, we report that acidic residues in the BAZ2B PHD domain are essential for H3 binding and that BAZ2B PHD–BRD establishes a polyvalent interaction with H3K14ac. Furthermore, we provide evidence that the disordered interdomain linker modulates the histone-binding affinity by interacting with the PHD domain. In particular, lysine-rich stretches in the linker, which resemble the positively charged N terminus of histone H3, reduce the binding affinity of the PHD finger toward the histone substrate. Phosphorylation, acetylation, or poly(ADP-ribosyl)ation of the linker residues may therefore act as a cellular mechanism to transiently tune BAZ2B histone-binding affinity. Our findings further support the concept of interdomain linkers serving a dual role in substrate binding by appropriately positioning the adjacent domains and by electrostatically modulating substrate binding. Moreover, inhibition of histone binding by a histone-mimicking interdomain linker represents another example of regulation of protein–protein interactions by intramolecular mimicry.

An Embryonic Stem Cell-Specific NuRD Complex Functions through Interaction with WDR5.

SummaryThe Nucleosome Remodeling and Deacetylase (NuRD) complex is a chromatin regulatory complex that functions as a transcriptional co-repressor in metazoans. The NuRD subunit MBD3 is essential for targeting and assembly of a functional NuRD complex as well as embryonic stem cell (ESC) pluripotency. Three MBD3 isoforms (MBD3A, MBD3B, and MBD3C) are expressed in mouse. Here, we find that the MBD3C isoform contains a unique 50-amino-acid N-terminal region that is necessary for MBD3C to specifically interact with the histone H3 binding protein WDR5. Domain analyses of WDR5 reveal that the H3 binding pocket is required for interaction with MBD3C. We find that while Mbd3c knockout ESCs differentiate normally, MBD3C is redundant with the MBD3A and MBD3B isoforms in regulation of gene expression, with the unique MBD3C N terminus required for this redundancy. Together, our data characterize a unique NuRD complex variant that functions specifically in ESCs.

Variations on a nucleosome theme: The structural basis of centromere function.

The centromere is a specialized chromosomal structure that dictates kinetochore assembly and, thus, is essential for accurate chromosome segregation. Centromere identity is determined epigenetically by the presence of a centromere-specific histone H3 variant, CENP-A, that replaces canonical H3 in centromeric chromatin. Here, we discuss recent work by Roulland et al. that identifies structural elements of the nucleosome as essential determinants of centromere function. In particular, CENP-A nucleosomes have flexible DNA ends due to the short αN helix of CENP-A. The higher flexibility of the DNA ends of centromeric nucleosomes impairs binding of linker histones H1, while it facilitates binding of other essential centromeric proteins, such as CENP-C, and is required for mitotic fidelity. This work extends previous observations indicating that the differential structural properties of CENP-A nucleosomes are on the basis of its contribution to centromere identity and function. Here, we discuss the implications of this work and the questions arising from it.

Down-regulation of Claudin-2 Expression and Proliferation by Epigenetic Inhibitors in Human Lung Adenocarcinoma A549 Cells

Claudin-2 is highly expressed in lung adenocarcinoma tissues and increases proliferation in adenocarcinoma cells. The chemicals that reduce claudin-2 expression may have anti-cancer effects, but such therapeutic medicines have not been developed. We found that azacitidine (AZA), a DNA methylation inhibitor, and trichostatin A (TSA) and sodium butyrate (NaB), histone deacetylase (HDAC) inhibitors, decrease claudin-2 levels. The effect of AZA was mediated by the inhibition of phosphorylated Akt and NF-κB. LY-294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and BAY 11-7082, an NF-κB inhibitor, decreased claudin-2 levels. The reporter activity of claudin-2 was decreased by AZA and LY-294002, which was blocked by the mutation in a putative NF-κB-binding site. NF-κB bound to the promoter region of claudin-2, which was inhibited by AZA and LY-294002. AZA is suggested to decrease the claudin-2 mRNA level mediated by the inhibition of a PI3K/Akt/NF-κB pathway. TSA and NaB did not change phosphorylated Akt and NF-κB levels. Furthermore, these inhibitors did not change the reporter activity of claudin-2 but decreased the stability of claudin-2 mRNA mediated by the elevation of miR-497 microRNA. The binding of histone H3 to the promoter region of miR-497 was inhibited by TSA and NaB, whereas that of claudin-2 was not. These results suggest that HDAC inhibitors decrease claudin-2 levels mediated by the elevation of miR-497 expression. Cell proliferation was additively decreased by AZA, TSA, and NaB, which was partially rescued by ectopic expression of claudin-2. We suggest that epigenetic inhibitors suppress the abnormal proliferation of lung adenocarcinoma cells highly expressing claudin-2.

Interplay between H1 and HMGN epigenetically regulates OLIG1&2 expression and oligodendrocyte differentiation.

An interplay between the nucleosome binding proteins H1 and HMGN is known to affect chromatin dynamics, but the biological significance of this interplay is still not clear. We find that during embryonic stem cell differentiation loss of HMGNs leads to down regulation of genes involved in neural differentiation, and that the transcription factor OLIG2 is a central node in the affected pathway. Loss of HMGNs affects the expression of OLIG2 as well as that of OLIG1, two transcription factors that are crucial for oligodendrocyte lineage specification and nerve myelination. Loss of HMGNs increases the chromatin binding of histone H1, thereby recruiting the histone methyltransferase EZH2 and elevating H3K27me3 levels, thus conferring a repressive epigenetic signature at Olig1&2 sites. Embryonic stem cells lacking HMGNs show reduced ability to differentiate towards the oligodendrocyte lineage, and mice lacking HMGNs show reduced oligodendrocyte count and decreased spinal cord myelination, and display related neurological phenotypes. Thus, the presence of HMGN proteins is required for proper expression of neural differentiation genes during embryonic stem cell differentiation. Specifically, we demonstrate that the dynamic interplay between HMGNs and H1 in chromatin epigenetically regulates the expression of OLIG1&2, thereby affecting oligodendrocyte development and myelination, and mouse behavior.

Karyopherin-β2 Recognition of a PY-NLS Variant that Lacks the Proline-Tyrosine Motif

Karyopherin-β2 or Transportin-1 binds proline-tyrosine nuclear localization signals (PY-NLSs) in its cargos. PY-NLSs are described by structural disorder, overall positive charge, and binding epitopes composed of an N-terminal hydrophobic or basic motif and a C-terminal R-X2-5P-Y motif. The N-terminal tail of histone H3 binds Kapβ2 with high affinity but does not contain a recognizable PY-NLS. The crystal structure of the Kapβ2-H3 tail shows residues 11-27 of H3 binding to the PY-NLS site of Kapβ2. H3residues 11TGGKAPRK18 bind the site for PY-NLS Epitope 1 (N-terminal hydrophobic/basic motif), which is most important for Kapβ2-binding. H3 residue Arg26 occupies the PY-NLS Epitope 2 position (usually arginine of R-X2-5P-Y) but PY-NLS Epitope 3 (proline-tyrosine motif) is missing in the H3 tail. Histone H3 thus provides an example of a PY-NLS variant with no proline-tyrosine or homologous proline-hydrophobic motif. The H3 tail uses a very strong Epitope 1 to compensate for loss of the often-conserved proline-tyrosine epitope.

Dual effect of histone H4 on prothrombin activation

Essentials Prothrombin converts slowly to thrombin upon interaction with histone H4. Histone H4 may also affect the reactivity of prothrombin toward factor Xa. Histone H4 enhances or inhibits activation by factor Xa depending on cofactor Va. The results reveal an unanticipated dual effect of histone H4 on prothrombin activation by factor Xa. Background Recent studies have documented the ability of prothrombin to convert to the mature protease thrombin upon interaction with histone H4. The effect is abrogated by mutation of the catalytic Ser and requires the Gla domain. Objectives To explore the effect of histone H4 on the reactivity of prothrombin to its physiological activator factor (F) Xa, free or assembled in the prothrombinase complex. Methods The effect of histone H4 on prothrombin activation by FXa and prothrombinase is studied with kinetic assays. The potential epitope of prothrombin recognizing histone H4 is explored with electrostatic calculations using recent crystal structures. Results and Conclusions Binding of histone H4 has a dual effect on prothrombin activation by FXa that is of mechanistic significance: it enhances the reaction > 10-fold in the absence of cofactor Va, but produces complete inhibition in the presence of cofactor. Histone H4 binding to prothrombin produces very slow autoactivation independent of the coagulation cascade and promotes slow thrombin generation by FXa in the absence of phospholipids. In addition, histone H4 has a rapid and drastic inhibitory effect on prothrombin activation by prothrombinase that is likely to dominate pathophysiology.

DNA (De)Methylation: The Passive Route to Naïvety?

Mouse pluripotent embryonic stem (ES) cells can exist in distinct yet interchangeable epigenetic states dictated by their culture environment. Previous reports have shown that naïve pluripotent cells grown in the presence of 2i are characterised by global DNA hypomethylation and changes in the abundance and distribution of histone modifications. New research provides insights regarding how this might be achieved.

The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity

CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. Invivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.

Determination of receptor occupancy in the presence of mass dose: [11C]GSK189254 PET imaging of histamine H3 receptor occupancy by PF-03654746

Measurements of drug occupancies using positron emission tomography (PET) can be biased if the radioligand concentration exceeds “tracer” levels. Negative bias would also arise in successive PET scans if clearance of the radioligand is slow, resulting in a carryover effect. We developed a method to (1) estimate the in vivo dissociation constant Kd of a radioligand from PET studies displaying a non-tracer carryover (NTCO) effect and (2) correct the NTCO bias in occupancy studies taking into account the plasma concentration of the radioligand and its in vivo Kd. This method was applied in a study of healthy human subjects with the histamine H3 receptor radioligand [11C]GSK189254 to measure the PK-occupancy relationship of the H3 antagonist PF-03654746. From three test/retest studies, [11C]GSK189254 Kd was estimated to be 9.5 ± 5.9 pM. Oral administration of 0.1 to 4 mg of PF-03654746 resulted in occupancy estimates of 71%–97% and 30%–93% at 3 and 24 h post-drug, respectively. NTCO correction adjusted the occupancy estimates by 0%–15%. Analysis of the relationship between corrected occupancies and PF-03654746 plasma levels indicated that PF-03654746 can fully occupy H3 binding sites (ROmax = 100%), and its IC50 was estimated to be 0.144 ± 0.010 ng/mL. The uncorrected IC50 was 26% higher.

Convergent Mechanistic Features between the Structurally Diverse N- and O-Methyltransferases: Glycine N-Methyltransferase and Catechol O-Methyltransferase.

Although an enormous and still growing number of biologically diverse methyltransferases have been reported and identified, a comprehensive understanding of the enzymatic methyl transfer mechanism is still lacking. Glycine N-methyltransferase (GNMT), a member of the family that acts on small metabolites as the substrate, catalyzes methyl transfer from S-adenosyl-l-methionine (AdoMet) to glycine to form S-adenosyl-l-homocysteine and sarcosine. We report primary carbon (12C/14C) and secondary (1H3/3H3) kinetic isotope effects at the transferred methyl group, together with 1H3/3H3 binding isotope effects for wild-type GNMT and a series of Tyr21 mutants. The data implicate a compaction effect in the methyl transfer step that is conferred by the protein structure. Furthermore, a remarkable similarity of properties is observed between GNMT and catechol O-methyltransferase, despite significant differences between these enzymes with regard to their active site structures and catalyzed reactions. We attribute these results to a catalytically relevant reduction in the methyl donor–acceptor distance that is dependent on a tyrosine side chain positioned behind the methyl-bearing sulfur of AdoMet.