Functional and Structural Insights into Human PPARα/δ/γ Subtype Selectivity of Bezafibrate, Fenofibric Acid, and Pemafibrate.

Among the agonists against three peroxisome proliferator-activated receptor (PPAR) subtypes, those against PPARα (fibrates) and PPARγ (glitazones) are currently used to treat dyslipidemia and type 2 diabetes, respectively, whereas PPARδ agonists are expected to be the next-generation metabolic disease drug. In addition, some dual/pan PPAR agonists are currently being investigated via clinical trials as one of the first curative drugs against nonalcoholic fatty liver disease (NAFLD). Because PPARα/δ/γ share considerable amino acid identity and three-dimensional structures, especially in ligand-binding domains (LBDs), clinically approved fibrates, such as bezafibrate, fenofibric acid, and pemafibrate, could also act on PPARδ/γ when used as anti-NAFLD drugs. Therefore, this study examined their PPARα/δ/γ selectivity using three independent assays-a dual luciferase-based GAL4 transactivation assay for COS-7 cells, time-resolved fluorescence resonance energy transfer-based coactivator recruitment assay, and circular dichroism spectroscopy-based thermostability assay. Although the efficacy and efficiency highly varied between agonists, assay types, and PPAR subtypes, the three fibrates, except fenofibric acid that did not affect PPARδ-mediated transactivation and coactivator recruitment, activated all PPAR subtypes in those assays. Furthermore, we aimed to obtain cocrystal structures of PPARδ/γ-LBD and the three fibrates via X-ray diffraction and versatile crystallization methods, which we recently used to obtain 34 structures of PPARα-LBD cocrystallized with 17 ligands, including the fibrates. We herein reveal five novel high-resolution structures of PPARδ/γ-bezafibrate, PPARγ-fenofibric acid, and PPARδ/γ-pemafibrate, thereby providing the molecular basis for their application beyond dyslipidemia treatment.

A High-Methionine Diet for One-Week Induces a High Accumulation of Methionine in the Cerebrospinal Fluid and Confers Bipolar Disorder-like Behavior in Mice.

Methionine (Met) is considered the most toxic amino acid in mammals. Here, we investigated biochemical and behavioral impacts of ad libitum one-week feeding of high-Met diets on mice. Adult male mice were fed the standard rodent diet that contained 0.44% Met (1×) or a diet containing 16 graded Met doses (1.2×-13×). High-Met diets for one-week induced a dose-dependent decrease in body weight and an increase in serum Met levels with a 2.55 mM peak (versus basal 53 µM) on the 12×Met diet. Total homocysteine (Hcy) levels were also upregulated while concentrations of other amino acids were almost maintained in serum. Similarly, levels of Met and Hcy (but not the other amino acids) were highly elevated in the cerebrospinal fluids of mice on the 10×Met diet; the Met levels were much higher than Hcy and the others. In a series of behavioral tests, mice on the 10×Met diet displayed increased anxiety and decreased traveled distances in an open-field test, increased activity to escape from water soaking and tail hanging, and normal learning/memory activity in a Y-maze test, which were reflections of negative/positive symptoms and normal cognitive function, respectively. These results indicate that high-Met ad libitum feeding even for a week can induce bipolar disorder-like disease models in mice.

Crystal Structures of the Human Peroxisome Proliferator-Activated Receptor (PPAR)α Ligand-Binding Domain in Complexes with a Series of Phenylpropanoic Acid Derivatives Generated by a Ligand-Exchange Soaking Method.

Peroxisome proliferator-activated receptor (PPAR)α, a member of the nuclear receptor family, is a transcription factor that regulates the expression of genes related to lipid metabolism in a ligand-dependent manner, and has attracted attention as a target for hypolipidemic drugs. We have been developing phenylpropaonic acid derivatives as PPARα-targeted drug candidates for the treatment of metabolic diseases. Recently, we have developed the "ligand-exchange soaking method," which crystallizes the recombinant PPARα ligand-binding domain (LBD) as a complex with intrinsic fatty acids derived from an expression host Escherichia (E.) coli and thereafter replaces them with other higher-affinity ligands by soaking. Here we applied this method for preparation of cocrystals of PPARα LBD with its ligands that have not been obtained with the conventional cocrystallization method. We revealed the high-resolution structures of the cocrystals of PPARα LBD and the three synthetic phenylpropaonic acid derivatives: TIPP-703, APHM19, and YN4pai, the latter two of which are the first observations. The overall structures of cocrystals obtained from the two methods are identical and illustrate the close interaction between these ligands and the surrounding amino acid residues of PPARα LBD. This ligand-exchange soaking method could be applicable to high throughput preparations of co-crystals with another subtype PPARδ LBD for high resolution X-ray crystallography, because it also crystallizes in complex with intrinsic fatty acid(s) while not in the apo-form.

Structural Basis for Anti-non-alcoholic Fatty Liver Disease and Diabetic Dyslipidemia Drug Saroglitazar as a PPAR α/γ Dual Agonist.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors that consist of three subtypes (α, γ, and ß/δ) with distinct functions and PPAR dual/pan agonists are expected to be the next generation of drugs for metabolic diseases. Saroglitazar is the first clinically approved PPARα/γ dual agonist for treatment of diabetic dyslipidemia and is currently in clinical trials to treat non-alcoholic fatty liver disease (NAFLD); however, the structural information of its interaction with PPARα/γ remains unknown. We recently revealed the high-resolution co-crystal structure of saroglitazar and the PPARα-ligand binding domain (LBD) through X-ray crystallography, and in this study, we report the structure of saroglitazar and the PPARγ-LBD. Saroglitazar was located at the center of "Y"-shaped PPARγ-ligand-binding pocket (LBP), just as it was in the respective region of PPARα-LBP. Its carboxylic acid was attached to four amino acids (Ser289/His323/His449/Thr473), which contributes to the stabilization of Activating Function-2 helix 12, and its phenylpyrrole moiety was rotated 121.8 degrees in PPARγ-LBD from that in PPARα-LBD to interact with Phe264. PPARδ-LBD has the consensus four amino acids (Thr253/His287/His413/Tyr437) towards the carboxylic acids of its ligands, but it seems to lack sufficient space to accept saroglitazar because of the steric hindrance between the Trp228 or Arg248 residue of PPARδ-LBD and its methylthiophenyl moiety. Accordingly, in a coactivator recruitment assay, saroglitazar activated PPARα-LBD and PPARγ-LBD but not PPARδ-LBD, whereas glycine substitution of either Trp228, Arg248, or both of PPARδ-LBD conferred saroglitazar concentration-dependent activation. Our findings may be valuable in the molecular design of PPARα/γ dual or PPARα/γ/δ pan agonists.

Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria.

Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H2S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine ß-synthase (Cbs), cystathionine γ-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.

Abnormal Amino Acid Profiles of Blood and Cerebrospinal Fluid from Cystathionine ß-Synthase-Deficient Mice, an Animal Model of Homocystinuria.

Mental retardation is the most common feature among inborn errors of amino acid metabolism. Patients with homocystinuria/homocysteinemia caused by cystathionine ß-synthase (CBS) deficiency suffer from thromboembolism and mental retardation from early ages; therefore, detection by newborn screening is performed. Furthermore, elevated levels of serum homocysteine during pregnancy are associated with the occurrence of neural tube defects (NTDs) in newborns. However, the causes of such central nervous system (CNS) defects are unknown. We found previously impaired learning abilities in Cbs-deficient (Cbs-/-) mice (but not NTD births). Here, we investigated the amino acid profiles of serum and cerebrospinal fluid (CSF) from Cbs-/- mice. Mice deficient in cystathionine γ-lyase (Cth), a downstream enzyme of CBS in transsulfuration, as well as wild-type mice, were analyzed as controls. Cbs-/- and Cth-/- mice were smaller than wild-type mice, and CSF yields in Cbs-/- mice were lower than the others. CSF amino acid levels were generally lower than those in serum, and compared with the dramatic amino acid level alterations in Cbs-/- mouse serum, alterations in CSF were less apparent. However, marked upregulation (versus wild-type) of aspartic acid/asparagine (Asp/Asn), glutamine (Gln), serine (Ser), threonine (Thr), phenylalanine (Phe), tyrosine (Tyr), methionine (Met), total homocysteine, and citrulline, and downregulation of lysine (Lys) were found in Cbs-/- mouse CSF. Because similar regulation of total homocysteine/citrulline/Lys was observed in the CSF of Cth-/- mice, which are free of CNS dysfunction, the reduced CSF volumes and the level changes of other amino acids could be relevant to Cbs-/--specific CNS defects.

Preeclampsia-Like Features and Partial Lactation Failure in Mice Lacking Cystathionine γ-Lyase-An Animal Model of Cystathioninuria.

Elevated plasma homocysteine levels are considered as a risk factor for cardiovascular diseases as well as preeclampsia-a pregnancy disorder characterized by hypertension and proteinuria. We previously generated mice lacking cystathionine γ-lyase (Cth) as cystathioninuria models and found them to be with cystathioninemia/homocysteinemia. We investigated whether Cth-deficient (Cth-/-) pregnant mice display any features of preeclampsia. Cth-/- females developed normally but showed mild hypertension (~10 mmHg systolic blood pressure elevation) in late pregnancy and mild proteinuria throughout development/pregnancy. Cth-/- dams had normal numbers of pups and exhibited normal maternal behavior except slightly lower breastfeeding activity. However, half of them could not raise their pups owing to defective lactation; they could produce/store the first milk in their mammary glands but not often provide milk to their pups after the first ejection. The serum oxytocin levels and oxytocin receptor expression in the mammary glands were comparable between wild-type and Cth-/- dams, but the contraction responses of mammary gland myoepithelial cells to oxytocin were significantly lower in Cth-/- dams. The contraction responses to oxytocin were lower in uteruses isolated from Cth-/- mice. Our results suggest that elevated homocysteine or other unknown factors in preeclampsia-like Cth-/- dams interfere with oxytocin that regulates milk ejection reflex.

Hemizygosity of transsulfuration genes confers increased vulnerability against acetaminophen-induced hepatotoxicity in mice.

The key mechanism for acetaminophen hepatotoxicity is cytochrome P450 (CYP)-dependent formation of N-acetyl-p-benzoquinone imine, a potent electrophile that forms protein adducts. Previous studies revealed the fundamental role of glutathione, which binds to and detoxifies N-acetyl-p-benzoquinone imine. Glutathione is synthesized from cysteine in the liver, and N-acetylcysteine is used as a sole antidote for acetaminophen poisoning. Here, we evaluated the potential roles of transsulfuration enzymes essential for cysteine biosynthesis, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH), in acetaminophen hepatotoxicity using hemizygous (Cbs(+/-) or Cth(+/-)) and homozygous (Cth(-/-)) knockout mice. At 4 h after intraperitoneal acetaminophen injection, serum alanine aminotransferase levels were highly elevated in Cth(-/-) mice at 150 mg/kg dose, and also in Cbs(+/-) or Cth(+/-) mice at 250 mg/kg dose, which was associated with characteristic centrilobular hepatocyte oncosis. Hepatic glutathione was depleted while serum malondialdehyde accumulated in acetaminophen-injected Cth(-/-) mice but not wild-type mice, although glutamate-cysteine ligase (composed of catalytic [GCLC] and modifier [GCLM] subunits) became more activated in the livers of Cth(-/-) mice with lower Km values for Cys and Glu. Proteome analysis using fluorescent two-dimensional difference gel electrophoresis revealed 47 differentially expressed proteins after injection of 150 mg acetaminophen/kg into Cth(-/-) mice; the profiles were similar to 1000 mg acetaminophen/kg-treated wild-type mice. The prevalence of Cbs or Cth hemizygosity is estimated to be 1:200-300 population; therefore, the deletion or polymorphism of either transsulfuration gene may underlie idiosyncratic acetaminophen vulnerability along with the differences in Cyp, Gclc, and Gclm gene activities.

Formation of sulfur adducts of N-acetyl-p-benzoquinoneimine, an electrophilic metabolite of acetaminophen in vivo: participation of reactive persulfides.

While N-acetyl-p-benzoquinoneimine (NAPQI), an electrophilic metabolite of acetaminophen (APAP), has been found to undergo GSH conjugation associated with its detoxification, interaction of NAPQI with nucleophilic per- and polysulfides produced by cystathionine γ-lyase (CSE), cystathionine ß-synthase, and/or other enzymes is not known. In the present study, we found that sulfur adducts such as the NAPQIH2-SSSCys adduct and the NAPQIH2-SSG adduct are produced in biological samples of mice upon APAP exposure. Our in vitro experiments indicated that the formation of these novel APAP metabolites is, at least in part, attributable to the interaction of CysSSnSH produced by CSE and GSH persulfide with APAP-derived NAPQI.

Neutral aminoaciduria in cystathionine ß-synthase-deficient mice; an animal model of homocystinuria.

The kidney is one of the major loci for the expression of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH). While CBS-deficient (Cbs(-/-)) mice display homocysteinemia/methioninemia and severe growth retardation, and rarely survive beyond the first 4 wk, CTH-deficient (Cth(-/-)) mice show homocysteinemia/cystathioninemia but develop with no apparent abnormality. This study examined renal amino acid reabsorption in those mice. Although both 2-wk-old Cbs(-/-) and Cth(-/-) mice had normal renal architecture, their serum/urinary amino acid profiles largely differed from wild-type mice. The most striking feature was marked accumulation of Met and cystathionine in serum/urine/kidney samples of Cbs(-/-) and Cth(-/-) mice, respectively. Levels of some neutral amino acids (Val, Leu, Ile, and Tyr) that were not elevated in Cbs(-/-) serum were highly elevated in Cbs(-/-) urine, and urinary excretion of other neutral amino acids (except Met) was much higher than expected from their serum levels, demonstrating neutral aminoaciduria in Cbs(-/-) (not Cth(-/-)) mice. Because the bulk of neutral amino acids is absorbed via a B(0)AT1 transporter and Met has the highest substrate affinity for B(0)AT1 than other neutral amino acids, hypermethioninemia may cause hyperexcretion of neutral amino acids.

Different Coactivator Recruitment to Human PPARα/δ/γ Ligand-Binding Domains by Eight PPAR Agonists to Treat Nonalcoholic Fatty Liver Disease.

Three peroxisome proliferator-activated receptor subtypes, PPARα, PPAR(ß/)δ, and PPARγ, exert ligand-dependent transcriptional control in concert with retinoid X receptors (RXRs) on various gene sets harboring PPAR response elements (PPREs) in their promoter regions. Ligand-bound PPAR/RXR complexes do not directly regulate transcription; instead, they recruit multiprotein coactivator complexes to specific genomic regulatory loci to cooperatively activate gene transcription. Several coactivators are expressed in a single cell; however, a ligand-bound PPAR can be associated with only one coactivator through a consensus LXXLL motif. Therefore, altered gene transcription induced by PPAR subtypes/agonists may be attributed to the recruitment of various coactivator species. Using a time-resolved fluorescence resonance energy transfer assay, we analyzed the recruitment of four coactivator peptides (PGC1α, CBP, SRC1, and TRAP220) to human PPARα/δ/γ-ligand-binding domains (LBDs) using eight PPAR dual/pan agonists (bezafibrate, fenofibric acid, pemafibrate, pioglitazone, elafibranor, lanifibranor, saroglitazar, and seladelpar) that are/were anticipated to treat nonalcoholic fatty liver disease. These agonists all recruited four coactivators to PPARα/γ-LBD with varying potencies and efficacy. Only five agonists (bezafibrate, pemafibrate, elafibranor, lanifibranor, and seladelpar) recruited all four coactivators to PPARδ-LBD, and their concentration-dependent responses differed from those of PPARα/γ-LBD. These results indicate that altered gene expression through consensus PPREs by different PPAR subtypes/agonists may be caused, in part, by different coactivators, which may be responsible for the unique pharmacological properties of these PPAR agonists.

2D-DIGE Proteomic Analysis of Mouse Liver Within 1 Week.

Several years have passed since LC (liquid chromatography)-MS (mass spectrometry) became the mainstream for proteomic analysis; however, conventional fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) continues to be an important technology that enables rapid and direct visualization of hundreds to thousands of proteins and their quantitative analyses. We can get global proteomic views using 2D-DIGE within 3 days and then identify proteins with differential expression levels using MALDI-TOF/MS and MASCOT search engine. Here, we describe our routine 2D-DIGE proteomic analysis of the liver isolated from mice in pathological conditions within 1 week.

Current Clinical Trial Status and Future Prospects of PPAR-Targeted Drugs for Treating Nonalcoholic Fatty Liver Disease.

The number of patients with nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) is increasing globally and is raising serious concerns regarding the increasing medical and economic burden incurred for their treatment. The progression of NASH to more severe conditions such as cirrhosis and hepatocellular carcinoma requires liver transplantation to avoid death. Therefore, therapeutic intervention is required in the NASH stage, although no therapeutic drugs are currently available for this. Several anti-NASH candidate drugs have been developed that enable treatment via the modulation of distinct signaling cascades and include a series of drugs targeting peroxisome proliferator-activated receptor (PPAR) subtypes (PPARα/δ/γ) that are considered to be attractive because they can regulate both systemic lipid metabolism and inflammation. Multiple PPAR dual/pan agonists have been developed but only a few of them have been evaluated in clinical trials for NAFLD/NASH. Herein, we review the current clinical trial status and future prospects of PPAR-targeted drugs for treating NAFLD/NASH. In addition, we summarize our recent findings on the binding modes and the potencies/efficacies of several candidate PPAR dual/pan agonists to estimate their therapeutic potentials against NASH. Considering that the development of numerous PPAR dual/pan agonists has been abandoned because of their serious side effects, we also propose a repositioning of the already approved, safety-proven PPAR-targeted drugs against NAFLD/NASH.

Functional and Structural Insights into the Human PPARα/δ/γ Targeting Preferences of Anti-NASH Investigational Drugs, Lanifibranor, Seladelpar, and Elafibranor.

No therapeutic drugs are currently available for nonalcoholic steatohepatitis (NASH) that progresses from nonalcoholic fatty liver via oxidative stress-involved pathways. Three cognate peroxisome proliferator-activated receptor (PPAR) subtypes (PPARα/δ/γ) are considered as attractive targets. Although lanifibranor (PPARα/δ/γ pan agonist) and saroglitazar (PPARα/γ dual agonist) are currently under investigation in clinical trials for NASH, the development of seladelpar (PPARδ-selective agonist), elafibranor (PPARα/δ dual agonist), and many other dual/pan agonists has been discontinued due to serious side effects or little/no efficacies. This study aimed to obtain functional and structural insights into the potency, efficacy, and selectivity against PPARα/δ/γ of three current and past anti-NASH investigational drugs: lanifibranor, seladelpar, and elafibranor. Ligand activities were evaluated by three assays to detect different facets of the PPAR activation: transactivation assay, coactivator recruitment assay, and thermal stability assay. Seven high-resolution cocrystal structures (namely, those of the PPARα/δ/γ-ligand-binding domain (LBD)-lanifibranor, PPARα/δ/γ-LBD-seladelpar, and PPARα-LBD-elafibranor) were obtained through X-ray diffraction analyses, six of which represent the first deposit in the Protein Data Bank. Lanifibranor and seladelpar were found to bind to different regions of the PPARα/δ/γ-ligand-binding pockets and activated all PPAR subtypes with different potencies and efficacies in the three assays. In contrast, elafibranor induced transactivation and coactivator recruitment (not thermal stability) of all PPAR subtypes, but the PPARδ/γ-LBD-elafibranor cocrystals were not obtained. These results illustrate the highly variable PPARα/δ/γ activation profiles and binding modes of these PPAR ligands that define their pharmacological actions.

[PPARα-Ligand Binding Modes Revealed by X-ray Crystallography].

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors that consist of three subtypes (α, γ, and ß/δ) with distinct physiological functions and ligand recognition. PPARs regulate energy metabolism and therefore become therapeutic targets for various metabolic diseases. While PPARα agonists are used as anti-dyslipidemia drugs and PPARγ agonists as anti-type 2 diabetes drugs, PPAR dual/pan agonists (that acts on two or three subtypes) are expected to treat non-alcoholic steatohepatitis (NASH), pulmonary fibrosis, etc. Structural analyses of PPAR-ligand-binding domain (LBD)-ligand co-crystals using X-ray crystallography have been done mainly on PPARγ, in which ligand-free apocrystals were prepared; however, the information on PPARα-LBD and PPARδ-LBD is limited. Recently, we succeeded to obtain 34 novel co-crystal structures of PPARα-LBD and various PPARα ligands (including fibrates) using various co-crystallization techniques. This procedure is applicable to preparation of PPARδ-LBD co-crystals, and contributes to molecular design of new PPAR targeted drugs based on all three PPAR-LBD structures.

Preparation of co-crystals of human PPARα-LBD and ligand for high-resolution X-ray crystallography.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptor-type transcription factors with three subtypes (α, δ, and γ) that regulate cell differentiation and metabolism. Co-crystals of human PPARα-ligand-binding domain (LBD)-PPARα ligand for X-ray crystallography have been difficult to obtain. Recombinant human PPARα-LBD proteins contain intrinsic fatty acids (iFAs of Escherichia coli origin) and may be unstable without ligands during crystallization. To circumvent these limitations, we have successfully applied various crystallization techniques, including co-crystallization, cross-seeding, soaking, delipidation, and coactivator peptide supplementation. For complete details on the use and execution of this protocol, please refer to Kamata et al. (2020).

Cytotoxicity comparison of 35 developmental neurotoxicants in human induced pluripotent stem cells (iPSC), iPSC-derived neural progenitor cells, and transformed cell lines.

The Organization for Economic Co-operation and Development (OECD) test guideline 426 for developmental neurotoxicity (DNT) of industrial/environmental chemicals depends primarily on animal experimentation. This requirement raises various critical issues, such as high cost, long duration, the sacrifice of large numbers of animals, and interspecies differences. This study demonstrates an alternative protocol that is simple, quick, less expensive, and standardized to evaluate DNT of many chemicals using human induced pluripotent stem cells (iPSC) and their differentiation to neural progenitor cells (NPC). Initially, concentration-dependent cytotoxicity of 35 DNT chemicals, including industrial materials, insecticides, and clinical drugs, were compared among iPSC, NPC, and two transformed cells, Cos-7 and HepG2, using tetrazolium dye (MTS)-reducing colorimetric and ATP luciferase assays, and IC50 values were calculated. Next, inhibitory effects of the 14 representative chemicals (mainly insecticides) on iPSC differentiation to NPC were evaluated by measuring altered expression of neural differentiation and undifferentiation marker genes. Results show that both iPSC and NPC were much more sensitive to most DNT chemicals than the transformed cells, and 14 chemicals induced differential patterns of marker gene expression, highlighting the validity and utility of the protocol for evaluation and classification of DNT chemicals and preclinical DNT tests for safety assessment.

PPARα Ligand-Binding Domain Structures with Endogenous Fatty Acids and Fibrates.

Most triacylglycerol-lowering fibrates have been developed in the 1960s-1980s before their molecular target, peroxisome proliferator-activated receptor alpha (PPARα), was identified. Twenty-one ligand-bound PPARα structures have been deposited in the Protein Data Bank since 2001; however, binding modes of fibrates and physiological ligands remain unknown. Here we show thirty-four X-ray crystallographic structures of the PPARα ligand-binding domain, which are composed of a "Center" and four "Arm" regions, in complexes with five endogenous fatty acids, six fibrates in clinical use, and six synthetic PPARα agonists. High-resolution structural analyses, in combination with coactivator recruitment and thermostability assays, demonstrate that stearic and palmitic acids are presumably physiological ligands; coordination to Arm III is important for high PPARα potency/selectivity of pemafibrate and GW7647; and agonistic activities of four fibrates are enhanced by the partial agonist GW9662. These results renew our understanding of PPARα ligand recognition and contribute to the molecular design of next-generation PPAR-targeted drugs.

Dietary selenium deficiency or selenomethionine excess drastically alters organ selenium contents without altering the expression of most selenoproteins in mice.

Selenium is an essential trace element, and its deficiency can cause cardiomyopathy, arrhythmias and increased susceptibility to infection. Such clinical symptoms are considered primarily attributed to decreased expression of some of the 25 selenocysteine-containing selenoproteins in humans. Conversely, a selenium-excessive diet can cause acute poisoning and chronic symptoms with unknown mechanisms. To reveal the impact of selenium deficiency and excess on selenoprotein expression in vivo, mice (that possess 24 selenoproteins) were fed with selenium-deficient or selenomethionine-excessive diets for up to 4 weeks, and the expression levels of nine representative selenoproteins [glutathione peroxidase (Gpx) 1/2/3/4, thioredoxin reductase 1/2, deiodinase 1, and selenoprotein P/S] were measured in 10 organs (brain, heart, liver, lung, kidney, pancreas, spleen, testis, skeletal muscle and thymus). We observed a time-dependent decrease in the selenium content of most organs (except testis) of selenium-deficient mice but not in the expression levels of the nine selenoproteins, with the exceptions of Gpx1/2 in the heart/liver/kidney/pancreas/spleen and Gpx3 in the pancreas/spleen. Serum lipid peroxidation levels were up-regulated in response to Se deficiency because of the decreased expression/activity of Gpx3, a plasma-type Gpx. In contrast, a time-dependent increase was observed in the selenium content of all organs but not the expression levels of the nine selenoproteins in most organs of selenomethionine-excessive mice; however, markedly elevated protein-bound selenium levels were observed in the liver/kidney. These results suggest that the systemic response to selenium deficiency and selenomethionine excess involves the down-regulation of some selenoproteins such as Gpx1/Gpx3 and up-regulation of selenium-containing proteins (not selenoproteins), respectively.

Rapid 2D DIGE Proteomic Analysis of Mouse Liver.

Several years have passed since LC-MS(/MS) became the mainstream for proteomic analysis; however, conventional 2D DIGE (two-dimensional difference gel electrophoresis) continues to be an important technology that enables rapid and direct visualization of hundreds to thousands of proteins and their quantitative analyses. We can get global proteomic views using 2D DIGE within 3 days, and then identify proteins with differential expression levels using MALDI-TOF/MS and MASCOT search engine within a week. Here, we describe our routine 2D DIGE proteomic analysis of the liver isolated from mice in pathological conditions.