ABSTRACT
To ensure the physical interaction between a protein and its ligand, many techniques can be applied. One of them, isothermal titration calorimetry (ITC), measures the heat exchange between a forming molecular complex and its milieu. From this heat exchange, it is possible to acquire the thermodynamic parameters, the binding stoichiometry and the affinity constant (Ka) between the two interacting binding partners, which can then be used to determine the dissociation constant (Kd). We made use of ITC to determine the true Kd of melatonin for its putative receptor MT3, also known as the enzyme quinone reductase 2 (NQO2). In this chapter, we describe the step-by-step procedure for performing this experiment and extend it to 2-iodomelatonin, a melatonin derivative that was used in the initial identification and characterization of MT3. The dissociation constants of melatonin and 2-iodomelatonin toward NQO2 derived from these experiments are in line with data reported previously, albeit using alternative techniques.
Subject(s)
Melatonin , Quinone Reductases , Calorimetry/methods , Humans , Ligands , Melatonin/metabolism , Protein Binding , Quinone Reductases/metabolism , ThermodynamicsABSTRACT
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone which possesses a wide range of biological effects. The effects mediated by melatonin are in part attributed to the antioxidant properties of the molecule, which may act as scavenger of free radicals, and also to the binding of melatonin to its protein targets. For a long time, melatonin had been described as a ligand of a putative "receptor" present in the mammalian brain. Several studies were thus carried out with the goal of clarifying the nature of this melatonin "receptor," which led to the discovery of MT3 as the third melatonin binding site. This binding site was confirmed independently by several groups, and it was eventually demonstrated that MT3 was the enzyme quinone reductase 2 (NQO2). Among the different approaches used to validate that MT3 was indeed NQO2, the co-crystallization of NQO2 with melatonin was key in demonstrating the exact binding site and mode of melatonin to the enzyme and led to a clear understanding of the residues important for protein binding and inhibition. In this chapter, we described the details for the cloning, expression, and purification of the human enzyme NQO2. We also describe a detailed protocol for the crystallization of melatonin with this protein.
Subject(s)
Melatonin , Quinone Reductases , 5-Methoxytryptamine , Animals , Antioxidants , Cloning, Molecular , Crystallization , Humans , Ligands , Mammals/metabolism , Melatonin/metabolism , Quinone Reductases/genetics , Quinone Reductases/metabolism , Receptors, Melatonin/metabolism , X-RaysABSTRACT
Huntington's disease (HD) is the most common monogenic neurodegenerative disease and is fatal. CAG repeat expansions in mutant Huntingtin (mHTT) exon 1 encode for polyglutamine (polyQ) stretches and influence age of onset and disease severity, depending on their length. mHTT is more structured compared to wild-type (wt) HTT, resulting in a decreased N-terminal conformational flexibility. mHTT inflexibility may contribute to both gain of function toxicity, due to increased mHTT aggregation propensity, but also to loss of function phenotypes, due to decreased interactions with binding partners. High-throughput-screening techniques to identify mHTT flexibility states and potential flexibility modifying small molecules are currently lacking. Here, we propose a novel approach for identifying small molecules that restore mHTT's conformational flexibility in human patient fibroblasts. We have applied a well-established antibody-based time-resolved Förster resonance energy transfer (TR-FRET) immunoassay, which measures endogenous HTT flexibility using two validated HTT-specific antibodies, to a high-throughput screening platform. By performing a small-scale compound screen, we identified several small molecules that can partially rescue mHTT inflexibility, presumably by altering HTT post-translational modifications. Thus, we demonstrated that the HTT TR-FRET immunoassay can be miniaturized and applied to a compound screening workflow in patient cells. This automated assay can now be used in large screening campaigns to identify previously unknown HD drugs and drug targets.
Subject(s)
Huntington Disease , Neurodegenerative Diseases , Fluorescence Resonance Energy Transfer , Humans , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , Huntington Disease/genetics , Mutation , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolismABSTRACT
Parkinson's disease (PD) is a fatal neurodegenerative disorder that is primarily caused by the degeneration and loss of dopaminergic neurons of the substantia nigra in the ventral midbrain. Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of late-onset PD identified to date, with G2019S being the most frequent LRRK2 mutation, which is responsible for up to 1-2% of sporadic PD and up to 6% of familial PD cases. As no treatment is available for this devastating disease, developing new therapeutic strategies is of foremost importance. Cellular models are commonly used for testing novel potential neuroprotective compounds. However, current cellular PD models either lack physiological relevance to dopaminergic neurons or are too complex and costly for scaling up the production process and for screening purposes. In order to combine biological relevance and throughput, we have developed a PD model in Lund human mesencephalic (LUHMES) cell-derived dopaminergic neurons by overexpressing wild-type (WT) and G2019S LRRK2 proteins. We show that these cells can differentiate into dopaminergic-like neurons and that expression of mutant LRRK2 causes a range of different phenotypes, including reduced nuclear eccentricity, altered mitochondrial and lysosomal morphologies, and increased dopaminergic cell death. This model could be used to elucidate G2019S LRRK2-mediated dopaminergic neural dysfunction and to identify novel molecular targets for disease intervention. In addition, our model could be applied to high-throughput and phenotypic screenings for the identification of novel PD therapeutics.
Subject(s)
Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/physiology , Models, Biological , Parkinson Disease/metabolism , Codon , Dopaminergic Neurons/metabolism , Humans , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2/metabolismABSTRACT
N-ribosyldihydronicotinamide:quinone oxidoreductase 2 (NQO2/QR2, Enzyme Commission number 1.10.99.2) is a cytosolic enzyme, abundant in the liver and variably expressed in mammalian tissues. Cloned 30 years ago, it was characterized as a flavoenzyme catalyzing the reduction of quinones and pseudoquinones. To do so, it uses exclusively N-alkyl nicotinamide derivatives, without being able to recognize NADH, the reference hydrure donor compound, in contrast to its next of a kind, NAD(P)H:quinone oxidoreductase 1 (NQO1). For a long time both enzymes have been considered as key detoxifying enzymes in quinone metabolism, but more recent findings point to a more toxifying function of NQO2, particularly with respect to ortho-quinones. In fact, during the reduction of substrates, NQO2 generates fairly unstable intermediates that reoxidize immediately back to the original quinone, creating a futile cycle, the byproducts of which are deleterious reactive oxygen species. Beside this peculiarity, it is a target for numerous drugs and natural compounds such as melatonin, chloroquine, imiquimod, resveratrol, piceatannol, quercetin, and other flavonoids. Most of these enzyme-ligand interactions have been documented by numerous crystallographic studies, and now NQO2 is one of the best represented proteins in the structural biology database. Despite evidence for a causative role in several important diseases, the functional role of NQO2 remains poorly explored. In the present review, we aimed at detailing the main characteristics of NQO2 from a molecular pharmacology perspective. By drawing a clear border between facts and speculations, we hope to stimulate the future research toward a better understanding of this intriguing drug target. SIGNIFICANCE STATEMENT: Evidence is reviewed on the prevalent toxifying function of N-ribosyldihydronicotinamide:quinone oxidoreductase 2 while catalyzing the reduction of ortho-quinones such as dopamine quinone. The product of this reaction is unstable and generates a futile but harmful cycle (substrate/product/substrate) associated with reactive oxygen species generation.
Subject(s)
Quinone Reductases/metabolism , Quinones/metabolism , Animals , Humans , Liver/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Neuronal interleukin-34 (IL-34) promotes the expansion of microglia in the central nervous system-microglial activation and expansion are in turn implicated in the pathogenesis of Huntington's disease (HD). We thus examined whether the accumulation of an amyloidogenic exon-1 fragment of mutant huntingtin (mHTTx1) modulates the expression of IL-34 in dopaminergic neurons derived from a human embryonic stem cell line. We found that mHTTx1 aggregates induce IL-34 production selectively in post-mitotic neurons. Exposure of neurons to DNA damaging agents or the excitotoxin NMDA elicited similar results suggesting that IL-34 induction may be a general response to neuronal stress including the accumulation of misfolded mHTTx1. We further determined that knockdown or blocking the activity of IκB kinase beta (IKKß) prevented the aggregation of mHTTx1 and subsequent IL-34 production. While elevated IL-34 itself had no effect on the aggregation or the toxicity of mHTTx1 in neuronal culture, IL-34 expression in a rodent brain slice model with intact neuron-microglial networks exacerbated mHTTx1-induced degeneration of striatal medium-sized spiny neurons. Conversely, an inhibitor of the IL-34 receptor reduced microglial numbers and ameliorated mHTTx1-mediated neurodegeneration. Together, these findings uncover a novel function for IKKß/mHTTx1 interactions in regulating IL-34 production, and implicate a role for IL-34 in non-cell-autonomous, microglial-dependent neurodegeneration in HD.
Subject(s)
Huntington Disease/metabolism , Huntington Disease/pathology , I-kappa B Kinase/metabolism , Interleukins/metabolism , Animals , Cell Line , Corpus Striatum/metabolism , Exons , Humans , Huntingtin Protein/genetics , Huntingtin Protein/metabolism , I-kappa B Kinase/genetics , Interleukins/genetics , Microglia/metabolism , Microglia/pathology , Neurons/metabolism , Neurotoxins/metabolism , Primary Cell Culture , RatsABSTRACT
Many neurodegenerative diseases are linked to amyloid aggregation. In Huntington's disease (HD), neurotoxicity correlates with an increased aggregation propensity of a polyglutamine (polyQ) expansion in exon 1 of mutant huntingtin protein (mHtt). Here we establish how the domains flanking the polyQ tract shape the mHtt conformational landscape in vitro and in neurons. In vitro, the flanking domains have opposing effects on the conformation and stabilities of oligomers and amyloid fibrils. The N-terminal N17 promotes amyloid fibril formation, while the C-terminal Proline Rich Domain destabilizes fibrils and enhances oligomer formation. However, in neurons both domains act synergistically to engage protective chaperone and degradation pathways promoting mHtt proteostasis. Surprisingly, when proteotoxicity was assessed in rat corticostriatal brain slices, either flanking region alone sufficed to generate a neurotoxic conformation, while the polyQ tract alone exhibited minimal toxicity. Linking mHtt structural properties to its neuronal proteostasis should inform new strategies for neuroprotection in polyQ-expansion diseases.
Subject(s)
Huntington Disease/pathology , Mutant Proteins/genetics , Mutant Proteins/toxicity , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/toxicity , Nuclear Proteins/genetics , Nuclear Proteins/toxicity , Peptides , Animals , Huntingtin Protein , Mutant Proteins/chemistry , Nerve Tissue Proteins/chemistry , Nuclear Proteins/chemistry , Protein Conformation , Protein Multimerization , RatsABSTRACT
Glioblastoma multiforme (GBM) carries a poor prognosis and continues to lack effective treatments. Glioblastoma stem cells (GSCs) drive tumor formation, invasion, and drug resistance and, as such, are the focus of studies to identify new therapies for disease control. Here, we identify the involvement of IKK and NF-κB signaling in the maintenance of GSCs. Inhibition of this pathway impairs self-renewal as analyzed in tumorsphere formation and GBM expansion as analyzed in brain slice culture. Interestingly, both the canonical and non-canonical branches of the NF-κB pathway are shown to contribute to this phenotype. One source of NF-κB activation in GBM involves the TGF-ß/TAK1 signaling axis. Together, our results demonstrate a role for the NF-κB pathway in GSCs and provide a mechanistic basis for its potential as a therapeutic target in glioblastoma.
Subject(s)
Cell Self Renewal , I-kappa B Kinase/metabolism , NF-kappa B/metabolism , Neoplastic Stem Cells/metabolism , Signal Transduction , Animals , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Cell Line, Tumor , Glioblastoma/genetics , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , I-kappa B Kinase/genetics , MAP Kinase Kinase Kinases/metabolism , NF-kappa B/genetics , RNA Interference , Rats , Spheroids, Cellular/metabolism , Tissue Culture Techniques , Transforming Growth Factor beta/metabolismABSTRACT
Protein conformational diseases exhibit complex pathologies linked to numerous molecular defects. Aggregation of a disease-associated protein causes the misfolding and aggregation of other proteins, but how this interferes with diverse cellular pathways is unclear. Here, we show that aggregation of neurodegenerative disease-related proteins (polyglutamine, huntingtin, ataxin-1, and superoxide dismutase-1) inhibits clathrin-mediated endocytosis (CME) in mammalian cells by aggregate-driven sequestration of the major molecular chaperone heat shock cognate protein 70 (HSC70), which is required to drive multiple steps of CME. CME suppression was also phenocopied by HSC70 RNAi depletion and could be restored by conditionally increasing HSC70 abundance. Aggregation caused dysregulated AMPA receptor internalization and also inhibited CME in primary neurons expressing mutant huntingtin, showing direct relevance of our findings to the pathology in neurodegenerative diseases. We propose that aggregate-associated chaperone competition leads to both gain-of-function and loss-of-function phenotypes as chaperones become functionally depleted from multiple clients, leading to the decline of multiple cellular processes. The inherent properties of chaperones place them at risk, contributing to the complex pathologies of protein conformational diseases.
Subject(s)
Clathrin/metabolism , Endocytosis/physiology , Molecular Chaperones/metabolism , Neurons/metabolism , Protein Conformation , Proteins/chemistry , Animals , Antibody Specificity , Binding, Competitive/physiology , Cloning, Molecular , DNA Primers/genetics , Fluorescence Recovery After Photobleaching , Fluorescent Antibody Technique, Indirect , HSC70 Heat-Shock Proteins/genetics , Immunoblotting , Mice , Plasmids/genetics , RNA, Small Interfering/geneticsABSTRACT
Resveratrol (3,5,4'-trihydroxylstilbene) has been proposed to elicit a variety of positive health effects including protection against cancer and cardiovascular disease. The highest affinity target of resveratrol identified so far is the oxidoreductase enzyme quinone reductase 2 (QR2), which is believed to function in metabolic reduction and detoxification processes; however, evidence exists linking QR2 to the metabolic activation of quinones, which can lead to cell toxicity. Therefore, inhibition of QR2 by resveratrol may protect cells against reactive intermediates and eventually cancer. With the aim of identifying novel inhibitors of QR2, we designed, synthesized, and tested two generations of resveratrol analogue libraries for inhibition of QR2. In addition, X-ray crystal structures of six of the resveratrol analogues in the active site of QR2 were determined. Several novel inhibitors of QR2 were successfully identified as well as a compound that inhibits QR2 with a novel binding orientation.
Subject(s)
Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Quinone Reductases/antagonists & inhibitors , Stilbenes/chemistry , Anilides/chemistry , Catalytic Domain , Crystallography, X-Ray , Enzyme Activation/drug effects , Humans , Inhibitory Concentration 50 , Molecular Structure , Resveratrol , Small Molecule Libraries , Stilbenes/chemical synthesis , Stilbenes/pharmacologyABSTRACT
The cellular heat shock response (HSR) protects cells from toxicity associated with defective protein folding, and this pathway is widely viewed as a potential pharmacological target to treat neurodegenerative diseases linked to protein aggregation. Here we show that the HSR is not activated by mutant huntingtin (HTT) even in cells selected for the highest expression levels and for the presence of inclusion bodies containing aggregated protein. Surprisingly, HSR activation by HSF1 overexpression or by administration of a small molecule activator lowers the concentration threshold at which HTT forms inclusion bodies in cells expressing aggregation-prone, polyglutamine-expanded fragments of HTT. These data suggest that the HSR does not mitigate inclusion body formation.
Subject(s)
Heat-Shock Response , Huntington Disease/metabolism , Inclusion Bodies/metabolism , Models, Biological , DNA-Binding Proteins/metabolism , HEK293 Cells , Heat Shock Transcription Factors , Humans , Huntington Disease/pathology , Mutant Proteins/metabolism , Protein Binding , Transcription Factors/metabolismABSTRACT
Huntington's disease (HD) typifies a class of inherited neurodegenerative disorders in which a CAG expansion in a single gene leads to an extended polyglutamine tract and misfolding of the expressed protein, driving cumulative neural dysfunction and degeneration. HD is invariably fatal with symptoms that include progressive neuropsychiatric and cognitive impairments, and eventual motor disability. No curative therapies yet exist for HD and related polyglutamine diseases; therefore, substantial efforts have been made in the drug discovery field to identify potential drug and drug target candidates for disease-modifying treatment. In this context, we review here a range of early-stage screening approaches based in in vitro, cellular, and invertebrate models to identify pharmacological and genetic modifiers of polyglutamine aggregation and induced neurodegeneration. In addition, emerging technologies, including high-content analysis, three-dimensional culture models, and induced pluripotent stem cells are increasingly being incorporated into drug discovery screening pipelines for protein misfolding disorders. Together, these diverse screening strategies are generating novel and exciting new probes for understanding the disease process and for furthering development of therapeutic candidates for eventual testing in the clinical setting.
Subject(s)
Huntington Disease , Neurodegenerative Diseases , Peptides/metabolism , Proteostasis Deficiencies/complications , Animals , Disease Models, Animal , Humans , Huntingtin Protein , Huntington Disease/complications , Huntington Disease/etiology , Huntington Disease/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurodegenerative Diseases/etiology , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Peptides/genetics , Proteostasis Deficiencies/geneticsABSTRACT
Protein misfolding and aggregation are widely implicated in an increasing number of human diseases providing for new therapeutic opportunities targeting protein homeostasis (proteostasis). The cellular response to proteotoxicity is highly regulated by stress signaling pathways, molecular chaperones, transport and clearance machineries that function as a proteostasis network (PN) to protect the stability and functional properties of the proteome. Consequently, the PN is essential at the cellular and organismal level for development and lifespan. However, when challenged during aging, stress, and disease, the folding and clearance machineries can become compromised leading to both gain-of-function and loss-of-function proteinopathies. Here, we assess the role of small molecules that activate the heat shock response, the unfolded protein response, and clearance mechanisms to increase PN capacity and protect cellular proteostasis against proteotoxicity. We propose that this strategy to enhance cell stress pathways and chaperone activity establishes a cytoprotective state against misfolding and/or aggregation and represents a promising therapeutic avenue to prevent the cellular damage associated with the variety of protein conformational diseases.
Subject(s)
Heat-Shock Response , Molecular Targeted Therapy/methods , Proteasome Inhibitors/pharmacology , Protein Conformation , Proteins/metabolism , Autophagy/drug effects , Histone Deacetylase Inhibitors/pharmacology , Homeostasis , Humans , Molecular Chaperones/metabolism , Protein Folding , Proteins/chemistry , Proteostasis Deficiencies/drug therapy , Proteostasis Deficiencies/metabolismABSTRACT
Protein homeostasis (proteostasis) is essential for cellular and organismal health. Stress, aging and the chronic expression of misfolded proteins, however, challenge the proteostasis machinery and the vitality of the cell. Enhanced expression of molecular chaperones, regulated by heat shock transcription factor-1 (HSF-1), has been shown to restore proteostasis in a variety of conformational disease models, suggesting this mechanism as a promising therapeutic approach. We describe the results of a screen comprised of â¼900,000 small molecules that identified new classes of small-molecule proteostasis regulators that induce HSF-1-dependent chaperone expression and restore protein folding in multiple conformational disease models. These beneficial effects to proteome stability are mediated by HSF-1, FOXO, Nrf-2 and the chaperone machinery through mechanisms that are distinct from current known small-molecule activators of the heat shock response. We suggest that modulation of the proteostasis network by proteostasis regulators may be a promising therapeutic approach for the treatment of a variety of protein conformational diseases.
Subject(s)
Drug Evaluation, Preclinical , Molecular Chaperones/drug effects , Proteins/drug effects , Proteostasis Deficiencies/drug therapy , Transcription Factors/drug effects , Animals , Caenorhabditis elegans , Cell Line , DNA-Binding Proteins/drug effects , Forkhead Transcription Factors/drug effects , Heat Shock Transcription Factors , Homeostasis/drug effects , Humans , NF-E2-Related Factor 2/drug effects , Protein Conformation/drug effects , Proteins/chemistry , Proteins/physiology , RatsABSTRACT
Resveratrol has demonstrated cancer chemopreventive activity in animal models and some clinical trials are underway. In addition, resveratrol was shown to promote cell survival, increase lifespan and mimic caloric restriction, thereby improving health and survival of mice on high-calorie diet. All of these effects are potentially mediated by the pleiotropic interactions of resveratrol with different enzyme targets including COX-1 (cyclo-oxygenase-1) and COX-2, NAD+-dependent histone deacetylase SIRT1 (sirtuin 1) and QR2 (quinone reductase 2). Nonetheless, the health benefits elicited by resveratrol as a direct result of these interactions with molecular targets have been questioned, since it is rapidly and extensively metabolized to sulfate and glucuronide conjugates, resulting in low plasma concentrations. To help resolve these issues, we tested the ability of resveratrol and its metabolites to modulate the function of some known targets in vitro. In the present study, we have shown that COX-1, COX-2 and QR2 are potently inhibited by resveratrol, and that COX-1 and COX-2 are also inhibited by the resveratrol 4'-O-sulfate metabolite. We determined the X-ray structure of resveratrol bound to COX-1 and demonstrate that it occupies the COX active site similar to other NSAIDs (non-steroidal anti-inflammatory drugs). Finally, we have observed that resveratrol 3- and 4'-O-sulfate metabolites activate SIRT1 equipotently to resveratrol, but that activation is probably a substrate-dependent phenomenon with little in vivo relevance. Overall, the results of this study suggest that in vivo an interplay between resveratrol and its metabolites with different molecular targets may be responsible for the overall beneficial health effects previously attributed only to resveratrol itself.
Subject(s)
Stilbenes/metabolism , Stilbenes/pharmacology , Animals , Anticarcinogenic Agents/chemistry , Anticarcinogenic Agents/metabolism , Anticarcinogenic Agents/pharmacology , Catalytic Domain , Crystallography, X-Ray , Cyclooxygenase 1/chemistry , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/chemistry , Cyclooxygenase 2/metabolism , Cyclooxygenase 2 Inhibitors/chemistry , Cyclooxygenase 2 Inhibitors/metabolism , Cyclooxygenase 2 Inhibitors/pharmacology , Cyclooxygenase Inhibitors/chemistry , Cyclooxygenase Inhibitors/metabolism , Cyclooxygenase Inhibitors/pharmacology , Humans , In Vitro Techniques , Mice , Models, Molecular , Quinone Reductases/antagonists & inhibitors , Resveratrol , Sirtuin 1/metabolism , Stilbenes/chemistryABSTRACT
Melatonin exerts its biological effects through at least two transmembrane G-protein-coupled receptors, MT1 and MT2, and a lower-affinity cytosolic binding site, designated MT3. MT3 has recently been identified as QR2 (quinone reductase 2) (EC 1.10.99.2) which is of significance since it links the antioxidant effects of melatonin to a mechanism of action. Initially, QR2 was believed to function analogously to QR1 in protecting cells from highly reactive quinones. However, recent studies indicate that QR2 may actually transform certain quinone substrates into more highly reactive compounds capable of causing cellular damage. Therefore it is hypothesized that inhibition of QR2 in certain cases may lead to protection of cells against these highly reactive species. Since melatonin is known to inhibit QR2 activity, but its binding site and mode of inhibition are not known, we determined the mechanism of inhibition of QR2 by melatonin and a series of melatonin and 5-hydroxytryptamine (serotonin) analogues, and we determined the X-ray structures of melatonin and 2-iodomelatonin in complex with QR2 to between 1.5 and 1.8 A (1 A=0.1 nm) resolution. Finally, the thermodynamic binding constants for melatonin and 2-iodomelatonin were determined by ITC (isothermal titration calorimetry). The kinetic results indicate that melatonin is a competitive inhibitor against N-methyldihydronicotinamide (K(i)=7.2 microM) and uncompetitive against menadione (K(i)=92 microM), and the X-ray structures shows that melatonin binds in multiple orientations within the active sites of the QR2 dimer as opposed to an allosteric site. These results provide new insights into the binding mechanisms of melatonin and analogues to QR2.
Subject(s)
Melatonin/analogs & derivatives , Melatonin/metabolism , Quinone Reductases/metabolism , Binding Sites , Crystallography, X-Ray , Humans , Kinetics , Melatonin/chemistry , Models, Molecular , Molecular Structure , Protein Binding , Quinone Reductases/genetics , ThermodynamicsABSTRACT
Lycopene, the red pigment of the tomato, is under investigation for the chemoprevention of prostate cancer. Because dietary lycopene has been reported to concentrate in the human prostate, its uptake and subcellular localization were investigated in the controlled environment of cell culture using the human prostate cancer cell lines LNCaP, PC-3, and DU145. After 24 hours of incubation with 1.48 micromol/L lycopene, LNCaP cells accumulated 126.6 pmol lycopene/million cells, which was 2.5 times higher than PC-3 cells and 4.5 times higher than DU145 cells. Among these cell lines, only LNCaP cells express prostate-specific antigen and fully functional androgen receptor. Levels of prostate-specific antigen secreted into the incubation medium by LNCaP cells were reduced 55% as a result of lycopene treatment at 1.48 micromol/L. The binding of lycopene to the ligand-binding domain of the human androgen receptor was carried out, but lycopene was not found to be a ligand for this receptor. Next, subcellular fractionation of LNCaP cells exposed to lycopene was carried out using centrifugation and followed by liquid chromatography-tandem mass spectrometry quantitative analysis to determine the specific cellular locations of lycopene. The majority of lycopene (55%) was localized to the nuclear membranes, followed by 26% in nuclear matrix, and then 19% in microsomes. No lycopene was detected in the cytosol. These data suggest that the rapid uptake of lycopene by LNCaP cells might be facilitated by a receptor or binding protein and that lycopene is stored selectively in the nucleus of LNCaP cells.
Subject(s)
Anticarcinogenic Agents/metabolism , Carotenoids/metabolism , Cell Nucleus/metabolism , Prostatic Neoplasms/metabolism , Anticarcinogenic Agents/analysis , Binding Sites , Carotenoids/analysis , Cell Nucleus/chemistry , Chromatography, Liquid , Humans , Kinetics , Lycopene , Male , Mass Spectrometry , Prostatic Neoplasms/chemistry , Receptors, Androgen/metabolism , Time Factors , Tumor Cells, CulturedABSTRACT
Organophosphorus hydrolase detoxifies a broad range of organophosphate pesticides and the chemical warfare agents (CWAs) sarin and VX. Previously, rational genetic engineering produced OPH variants with 30-fold enhancements in the hydrolysis of CWA and their analogs. One interesting variant (H254R) in which the histidine at position 254 was changed to an arginine showed a 4-fold increase in the hydrolysis of demetonS (VX analog), a 14-fold decrease with paraoxon (an insecticide), and a 183-fold decrease with DFP (sarin analog). The three-dimensional structure of this enzyme at 1.9A resolution with the inhibitor, diethyl 4-methylbenzylphosphonate (EBP), revealed that the inhibitor did not bind at the active site, but bound exclusively into a well-defined surface pocket 12 A away from the active site. This structural feature was accompanied by non-competitive inhibition of paraoxon hydrolysis by EBP with H254R, in contrast to the native enzyme, which showed competitive inhibition. These parallel structure-function characteristics identify a functional, allosteric site on the surface of this enzyme.