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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.
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One of the major global health and welfare issues is the treatment of obesity and associated metabolic disorders, such as type 2 diabetes mellitus and nonalcoholic fatty liver disease. Obesity, caused by the excessive accumulation of triglycerides in adipose tissues, induces adipocyte dysfunction, followed by inflammation, in adipose tissues and lipotoxicity in nonadipose tissues. Several studies have shown that obesity and glucose homeostasis are influenced by sphingolipid mediators, including ceramide and sphingosine 1-phosphate (S1P). Cellular accumulation of ceramide impairs pancreatic ß-cell survival, confers insulin resistance in the liver and the skeletal muscle, and deteriorates adipose tissue inflammation via unknown molecular mechanisms. The roles of S1P are more complicated, because there are five cell-surface S1P receptors (S1PRs: S1P1-5) which have altered functions, different cellular expression patterns, and inapparent intracellular targets. Recent findings, including those by our group, support the notable concept that the pharmacological activation of S1P1 or S1P3 improves obesity and associated metabolic disorders, whereas that of S1P2 has the opposite effect. In addition, the regulation of S1P production by sphingosine kinase (SphK) is an essential factor affecting glucose homeostasis. This review summarizes the current knowledge on SphK/S1P/S1PR signaling in and against obesity, insulin resistance, and associated disorders.
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Diabetes Mellitus Tipo 2 , Resistência à Insulina , Lisofosfolipídeos , Esfingosina/análogos & derivados , Humanos , Obesidade , Ceramidas , Inflamação , Homeostase , GlucoseRESUMO
Hydrogen sulfide (H2S), synthesized by cystathionine gamma-lyase (Cth), contributes to the inflammatory response observed in sepsis. This study examines the effect of Cth-derived H2S in adhesion molecules on endothelial cells of vital organs in mice in a cecal ligation puncture (CLP)-induced model of sepsis, using two different and complementary approaches: Cth gene deletion and pharmacological inhibition. Our findings revealed a decreased level of H2S-synthesizing activity (via Cth) in both Cth-/- mice and PAG-treated wild-type (WT) mice following CLP-induced sepsis. Both treatment groups had reduced MPO activity and expression of chemokines (MCP-1 and MIP-2α), adhesion molecules (ICAM-1 and VCAM-1), ERK1/2 phosphorylation, and NF-κB in the liver and lung compared with in CLP-WT mice. Additionally, we found that PAG treatment in Cth-/- mice had no additional effect on the expression of ERK1/2 phosphorylation, NF-κB, or the production of chemokines and adhesion molecules in the liver and lung compared to Cth-/- mice following CLP-induced sepsis. The WT group with sepsis had an increased immunoreactivity of adhesion molecules on endothelial cells in the liver and lung than the WT sham-operated control. The Cth-/-, PAG-treated WT, and Cth-/- groups of mice showed decreased immunoreactivity of adhesion molecules on endothelial cells in the liver and lung following sepsis. Inhibition of H2S production via both approaches reduced adhesion molecule expression on endothelial cells and reduced liver and lung injury in mice with sepsis. In conclusion, this study demonstrates that H2S has an important role in the pathogenesis of sepsis and validates PAG use as a suited tool for investigating the Cth/H2S-signalling axis in sepsis.
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Cistationina gama-Liase , Sepse , Animais , Camundongos , Moléculas de Adesão Celular , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/genética , Células Endoteliais , Deleção de Genes , NF-kappa B , Sepse/tratamento farmacológico , Sepse/genéticaRESUMO
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.
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Carcinoma Hepatocelular , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Humanos , Hipoglicemiantes , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , PPAR alfa , Ensaios Clínicos como AssuntoRESUMO
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.
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Reactive sulfur species, or persulfides and polysulfides, such as cysteine hydropersulfide and glutathione persulfide, are endogenously produced in abundance in both prokaryotes and eukaryotes, including mammals. Various forms of reactive persulfides occur in both low-molecular-weight and protein-bound thiols. The chemical properties and great supply of these molecular species suggest a pivotal role for reactive persulfides/polysulfides in different cellular regulatory processes (e.g., energy metabolism and redox signaling). We demonstrated earlier that cysteinyl-tRNA synthetase (CARS) is a new cysteine persulfide synthase (CPERS) and is responsible for the in vivo production of most reactive persulfides (polysulfides). Some researchers continue to suggest that 3-mercaptopyruvate sulfurtransferase (3-MST), cystathionine ß-synthase (CBS), and cystathionine γ-lyase (CSE) may also produce hydrogen sulfide and persulfides that may be generated during the transfer of sulfur from 3-mercaptopyruvate to the cysteine residues of 3-MST or direct synthesis from cysteine by CBS/CSE, respectively. We thus used integrated sulfur metabolome analysis, which we recently developed, with 3-MST knockout (KO) mice and CBS/CSE/3-MST triple-KO mice, to elucidate the possible contribution of 3-MST, CBS, and CSE to the production of reactive persulfides in vivo. We therefore quantified various sulfide metabolites in organs derived from these mutant mice and their wild-type littermates via this sulfur metabolome, which clearly revealed no significant difference between mutant mice and wild-type mice in terms of reactive persulfide production. This result indicates that 3-MST, CBS, and CSE are not major sources of endogenous reactive persulfide production; rather, CARS/CPERS is the principal enzyme that is actually involved in and even primarily responsible for the biosynthesis of reactive persulfides and polysulfides in vivo in mammals.
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The Special Issue "Amino Acid Metabolism and Regulation in Health and Disease 2 [...].
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AminoácidosRESUMO
Hydrogen sulfide (H2S) has been shown to act as both anti-inflammatory and pro-inflammatory mediators. Application of H2S donors generally protects against inflammation; however, experimental results using mice lacking endogenous H2S-producing enzymes, such as cystathionine γ-lyase (CTH) and mercaptopyruvate sulfurtransferase (MPST), are often contradictory. We herein examined two types of model hapten-induced inflammation models, colitis (an inflammatory bowel disease model of mucosal immunity) and contact dermatitis (a type IV allergic model of systemic immunity), in CTH-deficient (Cth-/-) and MPST-deficient (Mpst-/-) mice. Both mice exhibited no significant alteration from wild-type mice in trinitrobenzene sulfonic acid (Th1-type hapten)-induced colitis (a Crohn's disease model) and oxazolone (Th1/Th2 mix-type; Th2 dominant)-induced colitis (an ulcerative colitis model). However, Cth-/- (not Mpst-/-) mice displayed more exacerbated phenotypes in trinitrochlorobenzene (TNCB; Th1-type)-induced contact dermatitis, but not oxazolone, at the delayed phase (24 h post-administration) of inflammation. CTH mRNA expression was upregulated in the TNCB-treated ears of both wild-type and Mpst-/- mice. Although mRNA expression of pro-inflammatory cytokines (IL-1ß and IL-6) was upregulated in both early (2 h) and delayed phases of TNCB-triggered dermatitis in all genotypes, that of Th2 (IL-4) and Treg cytokines (IL-10) was upregulated only in Cth-/- mice, when that of Th1 cytokines (IFNγ and IL-2) was upregulated in wild-type and Mpst-/- mice at the delayed phase. These results suggest that (upregulated) CTH or H2S produced by it helps maintain Th1/Th2 balance to protect against contact dermatitis.
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Colite , Dermatite de Contato , Sulfeto de Hidrogênio , Camundongos , Animais , Cistationina gama-Liase/metabolismo , Sulfurtransferases/genética , Sulfeto de Hidrogênio/metabolismo , Colite/induzido quimicamente , Inflamação , Citocinas , Dermatite de Contato/etiologia , Haptenos , RNA Mensageiro , Cistationina beta-Sintase/metabolismoRESUMO
Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid that regulates fundamental cellular processes such as proliferation, migration, apoptosis, and differentiation through 5 cognate G protein-coupled receptors (S1P1-S1P5). We previously demonstrated that blockade of S1P2 signaling in S1P2-deficient mice attenuates high-fat diet-induced adipocyte hypertrophy and glucose intolerance and an S1P2-specific antagonist JTE-013 inhibits, whereas an S1P1/S1P3 dual antagonist (VPC23019) activates, adipogenic differentiation of preadipocytes. Based on those observations, this study examined whether an S1P1-specific agonist, SEW-2871, VPC23019, or their combination acts on obesity and glucose intolerance in leptin-deficient ob/ob mice. The oral administration of SEW-2871 or JTE-013 induced significant reductions in body/epididymal fat weight gains and epididymal/inguinal fat adipocyte sizes and improved glucose intolerance and adipocyte inflammation in ob/ob mice but not in their control C57BL/6J mice. Both SEW-2871 and JTE-013 decreased messenger RNA levels of tumor necrosis factor-α and CD11c, whereas they increased those of CD206 and adiponectin in the epididymal fats isolated from ob/ob mice with no changes in the levels of peroxisome proliferator activated receptor γ and its regulated genes. By contrast, VPC23019 did not cause any such alterations but counteracted with all those SEW-2871 actions in these mice. In conclusion, the S1P1 agonist SEW-2871 acted like the S1P2 antagonist JTE-013 to reduce body/epididymal fats and improve glucose tolerance in obese mice. Therefore, this study raises the possibility that endogenous S1P could promote obesity/type 2 diabetes through the S1P2, whereas exogenous S1P could act against them through the S1P1.
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Diabetes Mellitus Tipo 2 , Intolerância à Glucose , Animais , Masculino , Camundongos , Glucose , Lisofosfolipídeos/farmacologia , Lisofosfolipídeos/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade , Receptores de Lisoesfingolipídeo/genética , Esfingosina/farmacologia , Esfingosina/fisiologia , Receptores de Esfingosina-1-FosfatoRESUMO
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.
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Proteínas , Proteômica , Animais , Camundongos , Eletroforese em Gel Diferencial Bidimensional , Proteômica/métodos , Eletroforese em Gel Bidimensional/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas/metabolismo , Fígado/metabolismoRESUMO
Nonalcoholic fatty liver disease (NAFLD), including nonalcoholic fatty liver (NAFL) and a more advanced condition with inflammation/fibrosis, nonalcoholic steatohepatitis (NASH), is emerging as one of the most prevalent chronic diseases associated with the worldwide expansion of the obese population; however, there are currently only symptomatic therapy but no cure. Among multiple candidate drugs that have been developed and tried in clinical trials against NAFLD/NASH, peroxisome proliferator-activated receptor (PPAR) dual/pan agonists continue to be the most expected ones. This review summarizes the current condition of several PPAR agonists that were and are in clinical trials against NAFLD/NASH. In addition, we recently expanded structural information about PPARα/δ/γ-ligand interactions by X-ray crystallography and executed comparative functional analyses of PPARα/δ/γ activation by those ligands; based on those knowledge, we propose the reevaluation or repositioning of currently approved PPAR agonists, saroglitazar, bezafibrate, and pemafibrate, for the treatment of NAFLD/NASH.
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Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/etiologia , PPAR alfa , Hipoglicemiantes , Bezafibrato , ObesidadeRESUMO
Background Accumulating evidence suggests that hydrogen sulfide ( H2S ), an endogenously produced gaseous molecule, plays a critical role in the regulation of cardiovascular homeostasis. However, little is known about its role in lymphangiogenesis. Thus, the current study aimed to investigate the involvement of H2S in lymphatic vessel growth and lymphedema resolution using a murine model and assess the underlying mechanisms. Methods and Results A murine model of tail lymphedema was created both in wild-type mice and cystathionine γ-lyase-knockout mice, to evaluate lymphedema up to 28 days after lymphatic ablation. Cystathionine γ-lyase-knockout mice had greater tail diameters than wild-type mice, and this phenomenon was associated with the inhibition of reparative lymphangiogenesis at the site of lymphatic ablation. In contrast, the administration of an H2S donor, diallyl trisulfide, ameliorated lymphedema by inducing the formation of a considerable number of lymphatic vessels at the injured sites in the tails. In vitro experiments using human lymphatic endothelial cells revealed that diallyl trisulfide promoted their proliferation and differentiation into tube-like structures by enhancing Akt (protein kinase B) phosphorylation in a concentration-dependent manner. The blockade of Akt activation negated the diallyl trisulfide-induced prolymphangiogenic responses in lymphatic endothelial cells. Furthermore, the effects of diallyl trisulfide treatment on lymphangiogenesis in the tail lymphedema model were also negated by the inhibition of phosphoinositide 3'-kinase (P13K)/Akt signaling. Conclusions H2S promotes reparative lymphatic vessel growth and ameliorates secondary lymphedema, at least in part, through the activation of the Akt pathway in lymphatic endothelial cells. As such, H2S donors could be used as therapeutics against refractory secondary lymphedema.
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Sulfeto de Hidrogênio , Linfedema , Camundongos , Humanos , Animais , Linfangiogênese/fisiologia , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/metabolismo , Proteínas Proto-Oncogênicas c-akt , Fosfatidilinositol 3-Quinases , Cistationina gama-Liase/metabolismo , Células Endoteliais/metabolismo , Modelos Animais de Doenças , Linfedema/tratamento farmacológico , Camundongos KnockoutRESUMO
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.
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Diabetes Mellitus Tipo 2 , Dislipidemias , Hepatopatia Gordurosa não Alcoólica , PPAR delta , Benzoxazóis , Bezafibrato/farmacologia , Bezafibrato/uso terapêutico , Butiratos , Diabetes Mellitus Tipo 2/metabolismo , Dislipidemias/tratamento farmacológico , Fenofibrato/análogos & derivados , Humanos , Ligantes , PPAR alfa/metabolismo , PPAR delta/agonistas , PPAR gama/metabolismoRESUMO
Viral spike proteins play important roles in the viral entry process, facilitating attachment to cellular receptors and fusion of the viral envelope with the cell membrane. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor angiotensin converting enzyme-2 (ACE2) via its receptor-binding domain (RBD). The cysteine residue at position 488, consisting of a disulfide bridge with cysteine 480 is located in an important structural loop at ACE2-binding surface of RBD, and is highly conserved among SARS-related coronaviruses. We showed that the substitution of Cys-488 with alanine impaired pseudotyped SARS-CoV-2 infection, syncytium formation, and cell-cell fusion triggered by SARS-CoV-2 spike expression. Consistently, in vitro binding of RBD and ACE2, spike-mediated cell-cell fusion, and pseudotyped viral infection of VeroE6/TMPRSS2 cells were inhibited by the thiol-reactive compounds N-acetylcysteine (NAC) and a reduced form of glutathione (GSH). Furthermore, we demonstrated that the activity of variant spikes from the SARS-CoV-2 alpha and delta strains were also suppressed by NAC and GSH. Taken together, these data indicate that Cys-488 in spike RBD is required for SARS-CoV-2 spike functions and infectivity, and could be a target of anti-SARS-CoV-2 therapeutics.
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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.
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Transtorno Bipolar/psicologia , Homocisteína/sangue , Metionina/efeitos adversos , Aminoácidos/sangue , Aminoácidos/líquido cefalorraquidiano , Animais , Transtorno Bipolar/sangue , Transtorno Bipolar/líquido cefalorraquidiano , Transtorno Bipolar/induzido quimicamente , Modelos Animais de Doenças , Esquema de Medicação , Homocisteína/líquido cefalorraquidiano , Masculino , Metionina/sangue , Metionina/líquido cefalorraquidiano , Camundongos , Teste de Campo Aberto/efeitos dos fármacos , Regulação para CimaRESUMO
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.
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Cristalografia por Raios X/métodos , Ligantes , PPAR alfa/química , PPAR alfa/metabolismo , Metabolismo Energético , Hipoglicemiantes , Hipolipemiantes , Doenças Metabólicas/metabolismo , PPAR alfa/agonistas , PPAR alfa/fisiologia , Ligação Proteica , Domínios ProteicosRESUMO
Pathologic calcification (PC) is a painful and disabling condition whereby calcium-containing crystals deposit in tissues that do not physiologically calcify: cartilage, tendons, muscle, vessels and skin. In cartilage, compression and inflammation triggered by PC leads to cartilage degradation typical of osteoarthritis (OA). The PC process is poorly understood and treatments able to target the underlying mechanisms of the disease are lacking. Here we show a crucial role of the gasotransmitter hydrogen sulfide (H2S) and, in particular, of the H2S-producing enzyme cystathionine γ-lyase (CSE), in regulating PC in cartilage. Cse deficiency (Cse KO mice) exacerbated calcification in both surgically-induced (menisectomy) and spontaneous (aging) murine models of cartilage PC, and augmented PC was closely associated with cartilage degradation (OA). On the contrary, Cse overexpression (Cse tg mice) protected from these features. In vitro, Cse KO chondrocytes showed increased calcification, potentially via enhanced alkaline phosphatase (Alpl) expression and activity and increased IL-6 production. The opposite results were obtained in Cse tg chondrocytes. In cartilage samples from patients with OA, CSE expression inversely correlated with the degree of tissue calcification and disease severity. Increased cartilage degradation in murine and human tissues lacking or expressing low CSE levels may be accounted for by dysregulated catabolism. We found higher levels of matrix-degrading metalloproteases Mmp-3 and -13 in Cse KO chondrocytes, whereas the opposite results were obtained in Cse tg cells. Finally, by high-throughput screening, we identified a novel small molecule CSE positive allosteric modulator (PAM), and demonstrated that it was able to increase cellular H2S production, and decrease murine and human chondrocyte calcification and IL-6 secretion. Together, these data implicate impaired CSE-dependent H2S production by chondrocytes in the etiology of cartilage PC and worsening of secondary outcomes (OA). In this context, enhancing CSE expression and/or activity in chondrocytes could represent a potential strategy to inhibit PC.
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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.