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Lipoprotein-associated phospholipase A2 (Lp-PLA2) associates with low- and high-density lipoproteins in human plasma and specifically hydrolyzes circulating oxidized phospholipids involved in oxidative stress. The association of this enzyme with the lipoprotein's phospholipid monolayer to access its substrate is the most crucial first step in its catalytic cycle. The current study demonstrates unequivocally that a significant movement of a major helical peptide region occurs upon membrane binding, resulting in a large conformational change upon Lp-PLA2 binding to a phospholipid surface. This allosteric regulation of an enzyme's activity by a large membrane-like interface inducing a conformational change in the catalytic site defines a unique dimension of allosterism. The mechanism by which this enzyme associates with phospholipid interfaces to select and extract a single phospholipid substrate molecule and carry out catalysis is key to understanding its physiological functioning. A lipidomics platform was employed to determine the precise substrate specificity of human recombinant Lp-PLA2 and mutants. This study uniquely elucidates the association mechanism of this enzyme with membranes and its resulting conformational change as well as the extraction and binding of specific oxidized and short acyl-chain phospholipid substrates. Deuterium exchange mass spectrometry coupled with molecular dynamics simulations was used to define the precise specificity of the subsite for the oxidized fatty acid at the sn-2 position of the phospholipid backbone. Despite the existence of several crystal structures of this enzyme cocrystallized with inhibitors, little was understood about Lp-PLA2's specificity toward oxidized phospholipids.
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1-Alquil-2-acetilglicerofosfocolina Esterase/química , 1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Regulação Alostérica , Sítios de Ligação , Catálise , Domínio Catalítico , Ácidos Graxos , Humanos , Hidrólise , Lipoproteínas HDL/metabolismo , Membranas , Simulação de Dinâmica Molecular , Fosfolipídeos/metabolismo , Especificidade por SubstratoRESUMO
C14H20 (tetradecapentaene) is a simple model system exhibiting post transition-state behavior, wherein both the (6 + 4) and (4 + 2) cycloaddition products are formed from one ambimocal transition state structure. We studied the bifurcation dynamics starting from the two ambimodal transition state structures, the chair-form and boat-form, using the quasi-classical trajectory, classical molecular dynamics, and ring-polymer molecular dynamics methods on the parameter-optimized semiempirical GFN2-xTB potential energy surface. It was found that the calculated branching fractions differ between the chair-form and boat-form due to the different nature in the IRC pathways. We also investigated the effects of temperature on bifurcation dynamics and found that, at higher temperatures, trajectories stay longer in the intermediate region of the potential energy surface.
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Fibroblast accumulation and extracellular matrix (ECM) deposition are common critical steps for the progression of organ fibrosis, but the precise molecular mechanisms remain to be fully investigated. We have previously demonstrated that lysophosphatidic acid contributes to organ fibrosis through the production of connective tissue growth factor (CTGF) via actin cytoskeleton-dependent signaling, myocardin-related transcription factor family (MRTF) consisting of MRTF-A and MRTF-B-serum response factor (SRF) pathway. In this study, we investigated the role of the MRTF-SRF pathway in the development of renal fibrosis, focusing on the regulation of ECM-focal adhesions (FA) in renal fibroblasts. Here we showed that both MRTF-A and -B were required for the expressions of ECM-related molecules such as lysyl oxidase family members, type I procollagen and fibronectin in response to transforming growth factor (TGF)-ß1 . TGF-ß1 -MRTF-SRF pathway induced the expressions of various components of FA such as integrin α subunits (αv , α2 , α11 ) and ß subunits (ß1 , ß3 , ß5 ) as well as integrin-linked kinase (ILK). On the other hand, the blockade of ILK suppressed TGF-ß1 -induced MRTF-SRF transcriptional activity, indicating a mutual relationship between MRTF-SRF and FA. Myofibroblast differentiation along with CTGF expression was also dependent on MRTF-SRF and FA components. Finally, global MRTF-A deficient and inducible fibroblast-specific MRTF-B deficient mice (MRTF-AKO BiFBKO mice) are protected from renal fibrosis with adenine administration. Renal expressions of ECM-FA components and CTGF as well as myofibroblast accumulation were suppressed in MRTF-AKO BiFBKO mice. These results suggest that the MRTF-SRF pathway might be a therapeutic target for renal fibrosis through the regulation of components forming ECM-FA in fibroblasts.
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Fibroblastos , Nefropatias , Fatores de Transcrição , Animais , Camundongos , Actinas/metabolismo , Fibroblastos/metabolismo , Fibrose , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Nefropatias/metabolismo , Nefropatias/patologiaRESUMO
AIM: Metabolic dysfunction is a risk factor for esophageal squamous cell carcinoma (ESCC). We investigated the impact of the recently proposed metabolic dysfunction-associated fatty liver disease (MAFLD) and its subtypes on ESCC recurrence after endoscopic treatment. METHODS: This multicenter observational cohort study enrolled consecutive patients newly diagnosed with ESCC after endoscopic treatment. Patients were classified into MAFLD or non-MAFLD groups. The MAFLD group was further classified into non-obese and obese MAFLD groups with a body mass index cutoff value of 25 kg/m2 . The impact of MAFLD on the recurrence of ESCC was evaluated using a decision tree algorithm and random forest analysis. RESULTS: A total of 147 patients (average age 69 years; male : female, 127:20; observational period, 2.4 years) were enrolled. The 1-, 3-, and 5-year recurrence rates were 2.0%, 21.1%, and 33.7%, respectively. Independent risk factors for the recurrence of ESCC were MAFLD (HR 2.2812; 95% confidence interval 1.0497-4.9571; p = 0.0373), drinking status, and smoking status. Metabolic dysfunction-associated fatty liver disease was identified as the second most important classifier for recurrence, followed by drinking status. The cumulative incidence of ESCC recurrence was higher in the MAFLD group than in the non-MAFLD group. In a subanalysis, the cumulative incidence of recurrence was significantly higher in the non-obese than in the obese MAFLD group among abstainers/non-drinkers. Directed acyclic graphs revealed that MAFLD directly contributes to ESCC recurrence. CONCLUSIONS: MAFLD was independently and directly associated with ESCC recurrence after endoscopic treatment; a high recurrence rate was observed in patients with non-obese MAFLD. Metabolic dysfunction-associated fatty liver disease may identify patients at high risk for ESCC recurrence.
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Alzheimer's disease is a neurologic disorder characterized by the accumulation of extracellular deposits of amyloid-ß (Aß) fibrils in the brain of patients. The key etiologic agent in Alzheimer's disease is not known; however oligomeric Aß appears detrimental to neuronal functions and increases Aß fibrils deposition. Previous research has shown that curcumin, a phenolic pigment of turmeric, has an effect on Aß assemblies, although the mechanism remains unclear. In this study, we demonstrate that curcumin disassembles pentameric oligomers made from synthetic Aß42 peptides (pentameric oAß42), using atomic force microscopy imaging followed by Gaussian analysis. Since curcumin shows keto-enol structural isomerism (tautomerism), the effect of keto-enol tautomerism on its disassembly was investigated. We have found that curcumin derivatives capable of keto-enol tautomerization also disassemble pentameric oAß42, while, a curcumin derivative incapable of tautomerization did not affect the integrity of pentameric oAß42. These experimental findings indicate that keto-enol tautomerism plays an essential role in the disassembly. We propose a mechanism for oAß42 disassembly by curcumin based on molecular dynamics calculations of the tautomerism. When curcumin and its derivatives bind to the hydrophobic regions of oAß42, the keto-form changes predominantly to the enol-form; this transition is associated with structural (twisting, planarization and rigidification) and potential energy changes that give curcumin enough force to act as a torsion molecular-spring that eventually disassembles pentameric oAß42. This proposed mechanism sheds new light on keto-enol tautomerism as a relevant chemical feature for designing such novel therapeutic drugs that target protein aggregation.
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Doença de Alzheimer , Curcumina , Humanos , Curcumina/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Amiloide/metabolismo , Fragmentos de Peptídeos/metabolismoRESUMO
The drastic increase in the number of patients with diabetes and its complications is a global issue. Diabetic nephropathy, the leading cause of chronic kidney disease, significantly affects patients' quality of life and medical expenses. Furthermore, there are limited drugs for treating diabetic nephropathy patients. Impaired lipid signaling, especially abnormal protein kinase C (PKC) activation by de novo-synthesized diacylglycerol (DG) under high blood glucose, is one of the causes of diabetic nephropathy. DG kinase (DGK) is an enzyme that phosphorylates DG and generates phosphatidic acid, i.e., DGK can inhibit PKC activation under diabetic conditions. Indeed, it has been proven that DGK activation ameliorates diabetic nephropathy. In this review, we summarize the involvement of PKC and DGK in diabetic nephropathy as therapeutic targets, and its mechanisms, by referring to our recent study.
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Diabetes Mellitus , Nefropatias Diabéticas , Humanos , Diacilglicerol Quinase/metabolismo , Diacilglicerol Quinase/uso terapêutico , Nefropatias Diabéticas/tratamento farmacológico , Nefropatias Diabéticas/metabolismo , Diglicerídeos , Glicemia , Qualidade de Vida , Ácidos Fosfatídicos/uso terapêutico , Proteína Quinase C/metabolismoRESUMO
Human phospholipase A2s (PLA2) constitute a superfamily of enzymes that hydrolyze the sn-2 acyl-chain of glycerophospholipids, producing lysophospholipids and free fatty acids. Each PLA2 enzyme type contributes to specific biological functions based on its expression, subcellular localization, and substrate specificity. Among the PLA2 superfamily, the cytosolic cPLA2 enzymes, calcium-independent iPLA2 enzymes, and secreted sPLA2 enzymes are implicated in many diseases, but a central issue is the preference for double-bond positions in polyunsaturated fatty acids (PUFAs) occupying the sn-2 position of membrane phospholipids. We demonstrate that each PLA2 has a unique preference between the specific omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and the omega-6 arachidonic acid (AA), which are the precursors of most proinflammatory and anti-inflammatory or resolving eicosanoids and related oxylipins. Surprisingly, we discovered that human cPLA2 selectively prefers AA, whereas iPLA2 prefers EPA, and sPLA2 prefers DHA as substrate. We determined the optimal binding of each phospholipid substrate in the active site of each PLA2 to explain these specificities. To investigate this, we utilized recently developed lipidomics-based LC-MS/MS and GC/MS assays to determine the sn-2 acyl chain specificity in mixtures of phospholipids. We performed µs timescale molecular dynamics (MD) simulations to reveal unique active site properties, especially how the precise hydrophobic cavity accommodation of the sn-2 acyl chain contributes to the stability of substrate binding and the specificity of each PLA2 for AA, EPA, or DHA. This study provides the first comprehensive picture of the unique substrate selectivity of each PLA2 for omega-3 and omega-6 fatty acids.
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Ácidos Graxos Ômega-3/metabolismo , Ácidos Graxos Ômega-6/metabolismo , Fosfolipases A2/metabolismo , Ácidos Graxos Ômega-3/química , Ácidos Graxos Ômega-6/química , Humanos , Interações Hidrofóbicas e Hidrofílicas , Fosfolipases A2/químicaRESUMO
Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells through nitric oxide (NO) signaling due to up-regulated inducible NO synthase (iNOS) as early as within 3 days. Here, we investigated mechanisms of VPA-promoted neuronal differentiation of ASCs concerning the NO-citrulline cycle, the metabolic cycle producing NO. Cultured rat ASCs were differentiated to mature neuronal cells rich in dendrites and expressing a neuronal marker by treatments with VPA at 2 mM for 3 days and subsequently with the neuronal induction medium for 2 h. Inhibitor (α-methyl-d, l-aspartic acid, MDLA) of arginosuccinate synthase (ASS), a key enzyme of the NO-citrulline cycle, abolishes intracellular NO increase and VPA-promoted neuronal differentiation in ASCs. l-Arginine, the substrate of iNOS, restores the promotion effect of VPA, being against MDLA. Immunocytochemistry showed that ASS and iNOS were increased in ASCs expressing neurofilament medium polypeptide (NeFM), a neuronal marker, by VPA and NIM synergistically. Real-time RT-PCR analysis showed that mRNAs of Ass and arginosuccinate lyase (Asl) in the NO-citrulline cycle were increased by VPA. Chromatin immunoprecipitation assay indicated that Ass and Asl were up-regulated by VPA through the acetylation of their associated histone. From these results, it was considered that VPA up-regulated the whole NO-citrulline cycle, which enabled continuous NO production by iNOS in large amounts for potent iNOS-NO signaling to promote neuronal differentiation of ASCs. This may also indicate a mechanism enabling short-lived NO to function conveniently as a potent signaling molecule that can disappear quickly after its role.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Citrulina/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Ácido Valproico/farmacologia , Tecido Adiposo/citologia , Animais , Arginina/farmacologia , Argininossuccinato Sintase/antagonistas & inibidores , Argininossuccinato Sintase/metabolismo , Inibidores Enzimáticos/farmacologia , Masculino , N-Metilaspartato/análogos & derivados , N-Metilaspartato/farmacologia , Neurônios/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Ratos Wistar , Regulação para Cima/efeitos dos fármacosRESUMO
Activating the genetic potential of Streptomyces strains to produce secondary metabolites can improve the production of useful biologically active compounds and facilitate the discovery of novel biologically active compounds. In this study, we found that Streptomyces lividans carrying the R440H mutation in rpoB, encoding the RNA polymerase beta subunit, grown in the presence of lincomycin at concentrations below the minimum inhibitory concentration (MIC) produced abundant amounts of actinorhodin and certain cryptic secondary metabolites despite culture conditions that restrict their production by the wild-type strain. The results indicate that lincomycin at concentrations below the MIC may strongly potentiate secondary metabolite production by Streptomyces strains carrying a specific rpoB mutation. In this study, we report an interesting phenomenon induced by combining the positive effects of certain rpoB mutations and concentration-dependent responses to lincomycin on secondary metabolism in S. lividans 66 and discuss the mechanisms and their applicability in exploring cryptic secondary metabolite production in streptomycetes.
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Lincomicina , Streptomyces lividans , Antibacterianos , RNA Polimerases Dirigidas por DNA , Mutação , Metabolismo Secundário , Streptomyces lividans/genéticaRESUMO
Valproic acid (VPA) remarkably promotes the differentiation of adipose tissue-derived stem cells (ASCs) to mature neuronal cells, enabling neuronal induction within only three days. Here, we investigated the involvement of NO-signaling in the VPA-promoted neuronal differentiation of ASCs as a possible mechanism. Cultured rat ASCs were differentiated to matured neuronal cells rich in dendrites and expressing ßIII-tubulin protein, a neuronal marker, by treatments with VPA at 2â¯mM for 3 days and subsequently with the neuronal induction medium (NIM) containing cAMP-elevating agents for 2â¯h. Increased intracellular NO was detected in neuronal cells differentiated from ASCs treated with VPA by a fluorescence NO-specific probe, diaminofluorescein-FM diacetate. However, a NO donor (NOC18) increased the incidence of neuronal cells only to a lesser extent than VPA, indicating the insufficiency of exogenous NO. RT-PCR analysis of ASCs treated with VPA showed increased mRNA expression of inducible nitric oxide synthase (iNOS) with the acetylation of its associated histone H3K9. iNOS inhibitors (1400â¯W and dexamethasone) or a soluble guanylate cyclase (sGC) inhibitor (ODQ) decreased the incidence of neuronal cells differentiated from ASCs treated with VPA. These inhibitors also decreased the mRNA expression of mature neuronal markers, neurofilament medium polypeptide (NeFM) and microtubule-associated protein 2 (MAP2), as well as ßIII-tubulin (TUBB3), to various extents. It was considered from these results that VPA promoted mature neuronal differentiation of ASCs through the iNOS-NO-sGC signaling pathway. This provided insights into the regulated neuronal differentiation of ASCs in clinical applications.
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Diferenciação Celular/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/metabolismo , Ácido Valproico/farmacologia , Tecido Adiposo/citologia , Animais , Óxido Nítrico Sintase Tipo II/metabolismo , RNA Mensageiro/metabolismo , Ratos Wistar , Guanilil Ciclase Solúvel/metabolismoRESUMO
Diabetic nephropathy (DN) is a diabetic vascular complication, and abnormal protein kinase C (PKC) activation from increased diacylglycerol (DG) production in diabetic hyperglycemia is one of the causes of DN. Diacylglycerol kinase (DGK) converts DG into phosphatidic acid. In other words, DGK can attenuate PKC activity by reducing the amount of DG. Recently, we reported that intraperitoneally administered d-α-tocopherol (vitamin E, αToc) induces an amelioration of DN in vivo through the activation of DGKα and the prevention of podocyte loss. However, the effect of the oral administration of αToc on DN in mice remains unknown. Here, we evaluated the effect of oral administration of αToc on DN and its molecular mechanism using streptozocin-induced diabetic mice. Consequently, the oral administration of αToc significantly ameliorated the symptoms of DN by preventing the loss of podocytes, and it was revealed that the inhibition of PKCï activity was involved in this amelioration.
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Nefropatias Diabéticas/tratamento farmacológico , alfa-Tocoferol/uso terapêutico , Administração Oral , Animais , Camundongos , Podócitos/citologia , Podócitos/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , alfa-Tocoferol/administração & dosagemRESUMO
Phospholipase A2's (PLA2's) constitute a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain on glycerophospholipids. We have previously reported that each PLA2 Type shows a unique substrate specificity for the molecular species it hydrolyzes, especially the acyl chain that is cleaved from the sn-2 position and to some extent the polar group. However, phosphatidylinositol (PI) and PI phosphates (PIPs) have not been as well studied as substrates as other phospholipids because the PIPs require adaptation of the standard analysis methods, but they are important in vivo. We determined the in vitro activity of the three major types of human PLA2's, namely the cytosolic (c), calcium-independent (i), and secreted (s) PLA2's toward PI, PI-4-phosphate (PI(4)P), and PI-4,5-bisphosphate (PI(4,5)P2). The in vitro assay revealed that Group IVA cPLA2 (GIVA cPLA2) showed relatively high activity toward PI and PI(4)P among the tested PLA2's; nevertheless, the highly hydrophilic headgroup disrupted the interaction between the lipid surface and the enzyme. GIVA cPLA2 and GVIA iPLA2 showed detectable activity toward PI(4,5)P2, but it appeared to be a poorer substrate for all of the PLA2's tested. Furthermore, molecular dynamics (MD) simulations demonstrated that Thr416 and Glu418 of GIVA cPLA2 contribute significantly to accommodating the hydrophilic head groups of PI and PI(4)P, which could explain some selectivity for PI and PI(4)P. These results indicated that GIVA cPLA2 can accommodate PI and PI(4)P in its active site and hydrolyze them, suggesting that the GIVA cPLA2 may best account for the PI and PIP hydrolysis in living cells.
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Fosfatos de Fosfatidilinositol , Fosfatidilinositóis , Humanos , Fosfatos de Fosfatidilinositol/metabolismo , Especificidade por Substrato , Fosfatidilinositóis/metabolismo , Fosfolipases A2/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , HidróliseRESUMO
Adult neurogenesis confers the hippocampus with unparalleled neural plasticity, essential for intricate cognitive functions. The specific influence of sparse newborn neurons (NBNs) in modulating neural activities and subsequently steering behavior, however, remains obscure. Using an engineered NBN-tetanus toxin mouse model (NBN-TeTX), we noninvasively silenced NBNs, elucidating their crucial role in impulse inhibition and cognitive flexibility as evidenced through Morris water maze reversal learning and Go/Nogo task in operant learning. Task-based functional MRI (tb-fMRI) paired with operant learning revealed dorsal hippocampal hyperactivation during the Nogo task in male NBN-TeTX mice, suggesting that hippocampal hyperexcitability might underlie the observed behavioral deficits. Additionally, resting-state fMRI (rs-fMRI) exhibited enhanced functional connectivity between the dorsal and ventral dentate gyrus following NBN silencing. Further investigations into the activities of PV+ interneurons and mossy cells highlighted the indispensability of NBNs in maintaining the hippocampal excitation/inhibition balance. Our findings emphasize that the neural plasticity driven by NBNs extensively modulates the hippocampus, sculpting inhibitory control and cognitive flexibility.
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Cognição , Neurônios , Masculino , Animais , Camundongos , Aprendizagem , Interneurônios , Transmissão SinápticaRESUMO
Despite treatment advances, acute kidney injury (AKI)-related mortality rates are still high in hospitalized adults, often due to sepsis. Sepsis and AKI could synergistically worsen the outcomes of critically ill patients. TLR4 signaling and mitochondrial antiviral signaling protein (MAVS) signaling are innate immune responses essential in kidney diseases, but their involvement in sepsis-associated AKI (SA-AKI) remains unclear. We studied the role of MAVS in kidney injury related to the TLR4 signaling pathway using a murine LPS-induced AKI model in wild-type and MAVS-knockout mice. We confirmed the importance of M1 macrophage in SA-AKI through in vivo assessment of inflammatory responses. The TLR4 signaling pathway was upregulated in activated bone marrow-derived macrophages, in which MAVS helped maintain the LPS-suppressed TLR4 mRNA level. MAVS regulated redox homeostasis via NADPH oxidase Nox2 and mitochondrial reverse electron transport in macrophages to alleviate the TLR4 signaling response to LPS. Hypoxia-inducible factor 1α (HIF-1α) and AP-1 were key regulators of TLR4 transcription and connected MAVS-dependent reactive oxygen species signaling with the TLR4 pathway. Inhibition of succinate dehydrogenase could partly reduce inflammation in LPS-treated bone marrow-derived macrophages without MAVS. These findings highlight the renoprotective role of MAVS in LPS-induced AKI by regulating reactive oxygen species generation-related genes and maintaining redox balance. Controlling redox homeostasis through MAVS signaling may be a promising therapy for SA-AKI.
Assuntos
Injúria Renal Aguda , Sepse , Humanos , Animais , Camundongos , Lipopolissacarídeos , Espécies Reativas de Oxigênio/metabolismo , Receptor 4 Toll-Like/metabolismo , Injúria Renal Aguda/induzido quimicamente , Injúria Renal Aguda/metabolismo , Sepse/metabolismoRESUMO
Background: d-alanine administration prevented kidney damage in a murine acute kidney injury model. Further data are needed on the influence of d-alanine on kidney function in humans. Objective: This study investigated the effects of d-alanine intake on amino acid metabolism and kidney function in healthy volunteers. Methods: This multicenter pilot study randomly assigned individuals from the general Japanese population to receive 3 g or 6 g of d-alanine intake per day for 7 d in a 1:1 ratio. The primary endpoint was the mean change in plasma and urine d-alanine levels from baseline to 7 d after intake. The secondary endpoints were mean changes in kidney function and other clinical factors. Safety was assessed by evaluating adverse events and clinical parameters. Results: We randomly assigned 24 participants to the 3-g (n = 12) and 6-g d-alanine (n = 12) groups. The mean baseline estimated glomerular filtration rate (eGFR) was 73 mL/min/1.73 m2. The mean plasma d-alanine concentration increased from baseline by 77.5 ± 34.3 and 192.1 ± 80.9 nmol/mL in the 3-g and 6-g d-alanine groups (both p < 0.0001), respectively, in a dose-dependent manner (between-group difference: 114.6 nmol/mL; 95% CI: 62.1-167.2; P = 0.0002). A similar increase was observed for the urine d-alanine to creatinine ratio. The mean eGFR was elevated by 5.7 ± 8.8 mL/min/1.73 m2 in the 6-g d-alanine group (P = 0.045) but did not significantly change in the 3-g d-alanine group. Nonserious adverse events were reported in 11 participants. Conclusions: d-alanine intake increased plasma and urine d-alanine levels and was well tolerated in participants with normal kidney function. These results will be useful in future trials investigating the effects of d-alanine intake on kidney disease progression in patients with chronic kidney disease.This trial was registered at the UMIN Clinical Trials Registry as UMIN000051466.
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Glycerophospholipids are major components of cell membranes and consist of a glycerol backbone esterified with one of over 30 unique fatty acids at each of the sn-1 and sn-2 positions. In addition, in some human cells and tissues as much as 20% of the glycerophospholipids contain a fatty alcohol rather than an ester in the sn-1 position, although it can also occur in the sn-2 position. The sn-3 position of the glycerol backbone contains a phosphodiester bond linked to one of more than 10 unique polar head-groups. Hence, humans contain thousands of unique individual molecular species of phospholipids given the heterogeneity of the sn-1 and sn-2 linkage and carbon chains and the sn-3 polar groups. Phospholipase A2 (PLA2) is a superfamily of enzymes that hydrolyze the sn-2 fatty acyl chain resulting in lyso-phospholipids and free fatty acids that then undergo further metabolism. PLA2's play a critical role in lipid-mediated biological responses and membrane phospholipid remodeling. Among the PLA2 enzymes, the Group VIA calcium-independent PLA2 (GVIA iPLA2), also referred to as PNPLA9, is a fascinating enzyme with broad substrate specificity and it is implicated in a wide variety of diseases. Especially notable, the GVIA iPLA2 is implicated in the sequelae of several neurodegenerative diseases termed "phospholipase A2-associated neurodegeneration" (PLAN) diseases. Despite many reports on the physiological role of the GVIA iPLA2, the molecular basis of its enzymatic specificity was unclear. Recently, we employed state-of-the-art lipidomics and molecular dynamics techniques to elucidate the detailed molecular basis of its substrate specificity and regulation. In this review, we summarize the molecular basis of the enzymatic action of GVIA iPLA2 and provide a perspective on future therapeutic strategies for PLAN diseases targeting GVIA iPLA2.
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Cálcio , Doenças Neurodegenerativas , Humanos , Cálcio/metabolismo , Glicerol , Fosfolipases A2/metabolismo , Fosfolipídeos/metabolismo , Glicerofosfolipídeos/metabolismo , Fosfolipases A2 Independentes de CálcioRESUMO
Glycerophospholipids are major components of cell membranes and have enormous variation in the composition of fatty acyl chains esterified on the sn-1 and sn-2 position as well as the polar head groups on the sn-3 position of the glycerol backbone. Phospholipase A2 (PLA2) enzymes constitute a superfamily of enzymes which play a critical role in metabolism and signal transduction by hydrolyzing the sn-2 acyl chains of glycerophospholipids. In human cell membranes, in addition to the conventional diester phospholipids, a significant amount is the sn-1 ether-linked phospholipids which play a critical role in numerous biological activities. However, precisely how PLA2s distinguish the sn-1 acyl chain linkage is not understood. In the present study, we expanded the technique of lipidomics to determine the unique in vitro specificity of three major human PLA2s, including Group IVA cytosolic cPLA2, Group VIA calcium-independent iPLA2, and Group V secreted sPLA2 toward the linkage at the sn-1 position. Interestingly, cPLA2 prefers sn-1 vinyl ether phospholipids known as plasmalogens over conventional ester phospholipids and the sn-1 alkyl ether phospholipids. iPLA2 showed similar activity toward vinyl ether and ester phospholipids at the sn-1 position. Surprisingly, sPLA2 preferred ester phospholipids over alkyl and vinyl ether phospholipids. By taking advantage of molecular dynamics simulations, we found that Trp30 in the sPLA2 active site dominates its specificity for diester phospholipids.
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Fosfolipases A2/genética , Éteres Fosfolipídicos/metabolismo , Fosfolipídeos/genética , Compostos de Vinila/metabolismo , Cálcio/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Citosol/metabolismo , Glicerofosfolipídeos/química , Glicerofosfolipídeos/metabolismo , Humanos , Hidrólise , Cinética , Fosfolipases A2/metabolismo , Fosfolipídeos/metabolismo , Especificidade por Substrato/genética , Compostos de Vinila/químicaRESUMO
The quest for novel agents to regulate the generation of prostaglandin E2 (PGE2) is of high importance because this eicosanoid is a key player in inflammatory diseases. We synthesized a series of N-acylated and N-alkylated 2-aminobenzothiazoles and related heterocycles (benzoxazoles and benzimidazoles) and evaluated their ability to suppress the cytokine-stimulated generation of PGE2 in rat mesangial cells. 2-Aminobenzothiazoles, either acylated by the 3-(naphthalen-2-yl)propanoyl moiety (GK510) or N-alkylated by a chain carrying a naphthalene (GK543) or a phenyl moiety (GK562) at a distance of three carbon atoms, stand out in inhibiting PGE2 generation, with EC50 values ranging from 118 nM to 177 nM. Both GK510 and GK543 exhibit in vivo anti-inflammatory activity greater than that of indomethacin. Thus, N-acylated or N-alkylated 2-aminobenzothiazoles are novel leads for the regulation of PGE2 formation.
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Dinoprostona , Indometacina , Animais , Anti-Inflamatórios/farmacologia , Prostaglandinas E , RatosRESUMO
It is generally recognized that the main function of α-tocopherol (αToc), which is the most active form of vitamin E, is its antioxidant effect, while non-antioxidant effects have also been reported. We previously found that αToc ameliorates diabetic nephropathy via diacylglycerol kinase alpha (DGKα) activation in vivo, and the activation was not related to the antioxidant effect. However, the underlying mechanism of how αToc activates DGKα have been enigmatic. We report that the membrane-bound 67 kDa laminin receptor (67LR), which has previously been shown to serve as a receptor for epigallocatechin gallate (EGCG), also contains a novel binding site for vitamin E, and its association with Vitamin E mediates DGKα activation by αToc. We employed hydrogen-deuterium exchange mass spectrometry (HDX/MS) and molecular dynamics (MD) simulations to identify the specific binding site of αToc on the 67LR and discovered the conformation of the specific hydrophobic pocket that accommodates αToc. Also, HDX/MS and MD simulations demonstrated the detailed binding of EGCG to a water-exposed hydrophilic site on 67LR, while in contrast αToc binds to a distinct hydrophobic site. We demonstrated that 67LR triggers an important signaling pathway mediating non-antioxidant effects of αToc, such as DGKα activation. This is the first evidence demonstrating a membrane receptor for αToc and one of the underlying mechanisms of a non-antioxidant function for αToc.
Assuntos
Catequina , Diacilglicerol Quinase , Diacilglicerol Quinase/metabolismo , Vitamina E/farmacologia , Receptores de Laminina/metabolismo , Catequina/farmacologia , alfa-Tocoferol , Antioxidantes/farmacologia , Sítios de LigaçãoRESUMO
Significant efforts have been made to ameliorate diabetic nephropathy (DN) by inhibiting protein kinase C. However, these efforts have not been successful in human trials, suggesting that novel therapeutic strategies are required. Thus far, it has been reported that green tea polyphenol epigallocatechin gallate (EGCg) improved albuminuria in DN in a human trial. Our previous study revealed that activation of diacylglycerol kinase α (DGKα) plays a crucial role in the amelioration of DN and that EGCg activates DGKα. Here, we investigated whether and how DGKα contributes to the amelioration of DN upon stimulation by EGCg by using streptozotocin-induced type 1 diabetic model mice. Our results revealed that EGCg ameliorated albuminuria in DN through DGKα in vivo, and methylated EGCg, which has higher absorption in the plasma improved albuminuria in DN effectively. Additionally, we showed that c-Src mediated EGCg-induced DGKα translocation and colocalized with the 67 kDa laminin receptor, which is an EGCg receptor. Furthermore, EGCg attenuated the loss of podocytes in DN by preventing a decrease in focal adhesion under high glucose conditions. Our results indicate that the DGKα pathway is an attractive therapeutic target and that activating this pathway is a novel strategy for treating DN.