Structural Mechanism of S-Adenosyl Methionine Binding to Catechol O-Methyltransferase

Methyltransferases possess a homologous domain that requires both a divalent metal cation and S-adenosyl-L-methionine (SAM) to catalyze its reactions. The kinetics of several methyltransferases has been well characterized; however, the details regarding their structural mechanisms have remained unclear to date. Using catechol O-methyltransferase (COMT) as a model, we perform discrete molecular dynamics and computational docking simulations to elucidate the initial stages of cofactor binding. We find that COMT binds SAM via an induced-fit mechanism, where SAM adopts a different docking pose in the absence of metal and substrate in comparison to the holoenzyme. Flexible modeling of the active site side-chains is essential for observing the lowest energy state in the apoenzyme; rigid docking tools are unable to recapitulate the pose unless the appropriate side-chain conformations are given a priori. From our docking results, we hypothesize that the metal reorients SAM in a conformation suitable for donating its methyl substituent to the recipient ligand. The proposed mechanism enables a general understanding of how divalent metal cations contribute to methyltransferase function

Serotonin-Induced Hypersensitivity via Inhibition of Catechol O-Methyltransferase Activity

Abstract The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself (K i  = 44 μM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for β2- and β3-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions

Differential Regulation of 6- and 7-Transmembrane Helix Variants of μ-Opioid Receptor in Response to Morphine Stimulation

The pharmacological effect of opioids originates, at the cellular level, by their interaction with the μ-opioid receptor (mOR) resulting in the regulation of voltage-gated Ca2+ channels and inwardly rectifying K+ channels that ultimately modulate the synaptic transmission. Recently, an alternative six trans-membrane helix isoform of mOR, (6TM-mOR) has been identified, but its function and signaling are still largely unknown. Here, we present the structural and functional mechanisms of 6TM-mOR signaling activity upon binding to morphine. Our data suggest that despite the similarity of binding modes of the alternative 6TM-mOR and the dominant seven trans-membrane helix variant (7TM-mOR), the interaction with morphine generates different dynamic responses in the two receptors, thus, promoting the activation of different mOR-specific signaling pathways. We characterize a series of 6TM-mOR-specific cellular responses, and observed that they are significantly different from those for 7TM-mOR. Morphine stimulation of 6TM-mOR does not promote a cellular cAMP response, while it increases the intracellular Ca2+ concentration and reduces the cellular K+ conductance. Our findings indicate that 6TM-mOR has a unique contribution to the cellular opioid responses. Therefore, it should be considered as a relevant target for the development of novel pharmacological tools and medical protocols involving the use of opioids

Structural and functional interactions between six-transmembrane mu-opioid receptors and beta(2)-adrenoreceptors modulate opioid signaling

The primary molecular target for clinically used opioids is the mu-opioid receptor (MOR). Besides the major seven-transmembrane (7TM) receptors, the MOR gene codes for alternatively spliced six-transmembrane (6TM) isoforms, the biological and clinical significance of which remains unclear. Here, we show that the otherwise exclusively intracellular localized 6TM-MOR translocates to the plasma membrane upon coexpression with beta(2)-adrenergic receptors (beta(2)-ARs) through an interaction with the fifth and sixth helices of beta(2)-AR. Coexpression of the two receptors in BE(2)-C neuroblastoma cells potentiates calcium responses to a 6TM-MOR ligand, and this calcium response is completely blocked by a selective beta(2)-antagonist in BE(2)-C cells, and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and beta(2)-AR ligands leads to substantial analgesic synergy and completely reverses opioid-induced hyperalgesia in rodent behavioral models. Together, our results provide evidence that the heterodimerization of 6TM-MOR with beta(2)-AR underlies a molecular mechanism for 6TM cellular signaling, presenting a unique functional responses to opioids. This signaling pathway may contribute to the hyperalgesic effects of opioids that can be efficiently blocked by beta(2)-AR antagonists, providing a new avenue for opioid therapy.Peer reviewe

Low Enzymatic Activity Haplotypes of the Human Catechol-O-Methyltransferase Gene: Enrichment for Marker SNPs

Catechol-O-methyltransferase (COMT) is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous (val158met) position, designated as low (LPS), average (APS), and high pain sensitive (HPS), are associated with experimental pain sensitivity and risk of developing chronic musculoskeletal pain conditions. APS and HPS haplotypes produce significant functional effects, coding for 3- and 20-fold reductions in COMT enzymatic activity, respectively. In the present study, we investigated whether additional minor single nucleotide polymorphisms (SNPs), accruing in 1 to 5% of the population, situated in the COMT transcript region contribute to haplotype-dependent enzymatic activity. Computer analysis of COMT ESTs showed that one synonymous minor SNP (rs769224) is linked to the APS haplotype and three minor SNPs (two synonymous: rs6267, rs740602 and one nonsynonymous: rs8192488) are linked to the HPS haplotype. Results from in silico and in vitro experiments revealed that inclusion of allelic variants of these minor SNPs in APS or HPS haplotypes did not modify COMT function at the level of mRNA folding, RNA transcription, protein translation, or enzymatic activity. These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation. Thus, both minor synonymous and nonsynonymous SNPs in the coding region are markers of functional APS and HPS haplotypes rather than independent contributors to COMT activity

Polymorphisms in the glucocorticoid receptor co-chaperone FKBP5 predict persistent musculoskeletal pain after traumatic stress exposure

Individual vulnerability factors influencing the function of the hypothalamic-pituitary-adrenal (HPA) axis may contribute to the risk of the development of persistent musculoskeletal pain after traumatic stress exposure. The objective of the study was to evaluate the association between polymorphisms in the gene encoding FK506 binding protein 51, FKBP5, a glucocorticoid receptor co-chaperone, and musculoskeletal pain severity six weeks after two common trauma exposures. The study included data from two prospective emergency department-based cohorts: a discovery cohort (n=949) of European Americans experiencing motor vehicle collision and a replication cohort of adult European American women experiencing sexual assault (n=53). DNA was collected from trauma survivors at the time of initial assessment. Overall pain and neck pain six weeks after trauma exposure were assessed using a 0–10 numeric rating scale. After adjustment for multiple comparisons, six FKBP5 polymorphisms showed significant association (minimum p <0.0001) with both overall and neck pain in the discovery cohort. The association of rs3800373, rs9380526, rs9394314, rs2817032, and rs2817040 with neck pain and/or overall pain six weeks after trauma was replicated in the sexual assault cohort, showing the same direction of the effect in each case. The results of this study indicate that genetic variants in FKBP5 influence the severity of musculoskeletal pain symptoms experienced during the weeks after motor vehicle collision and sexual assault. These results suggest that glucocorticoid pathways influence the development of persistent post-traumatic pain, and that such pathways may be a target of pharmacologic interventions aimed at improving recovery after trauma

Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary1. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da2,3. Using genome-wide linkage analyses, we discovered an association between nerve-injury–induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain

Large meta-analysis of genome-wide association studies identifies five loci for lean body mass

Lean body mass, consisting mostly of skeletal muscle, is important for healthy aging. We performed a genome-wide association study for whole body (20 cohorts of European ancestry with n = 38,292) and appendicular (arms and legs) lean body mass (n = 28,330) measured using dual energy X-ray absorptiometry or bioelectrical impedance analysis, adjusted for sex, age, height, and fat mass. Twenty-one single-nucleotide polymorphisms were significantly associated with lean body mass either genome wide (p < 5 x 10(-8)) or suggestively genome wide (p < 2.3 x 10(-6)). Replication in 63,475 (47,227 of European ancestry) individuals from 33 cohorts for whole body lean body mass and in 45,090 (42,360 of European ancestry) subjects from 25 cohorts for appendicular lean body mass was successful for five single-nucleotide polymorphisms in/ near HSD17B11, VCAN, ADAMTSL3, IRS1, and FTO for total lean body mass and for three single-nucleotide polymorphisms in/ near VCAN, ADAMTSL3, and IRS1 for appendicular lean body mass. Our findings provide new insight into the genetics of lean body mass

Serotonin-Induced Hypersensitivity via Inhibition of Catechol O-Methyltransferase Activity

Abstract The subcutaneous and systemic injection of serotonin reduces cutaneous and visceral pain thresholds and increases responses to noxious stimuli. Different subtypes of 5-hydroxytryptamine (5-HT) receptors are suggested to be associated with different types of pain responses. Here we show that serotonin also inhibits catechol O-methyltransferase (COMT), an enzyme that contributes to modultion the perception of pain, via non-competitive binding to the site bound by catechol substrates with a binding affinity comparable to the binding affinity of catechol itself (Ki = 44 μM). Using computational modeling, biochemical tests and cellular assays we show that serotonin actively competes with the methyl donor S-adenosyl-L-methionine (SAM) within the catalytic site. Binding of serotonin to the catalytic site inhibits the access of SAM, thus preventing methylation of COMT substrates. The results of in vivo animal studies show that serotonin-induced pain hypersensitivity in mice is reduced by either SAM pretreatment or by the combined administration of selective antagonists for β2- and β3-adrenergic receptors, which have been previously shown to mediate COMT-dependent pain signaling. Our results suggest that inhibition of COMT via serotonin binding contributes to pain hypersensitivity, providing additional strategies for the treatment of clinical pain conditions.</p