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1.
Food Chem ; 329: 126775, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32512387

RESUMO

Fish products are a promising source of collagen; however, these extracts are biochemically unstable. Acid-soluble collagen (ASC) was isolated from the skin of eleven fish species at various physiological temperatures (Tp). Structural features of these samples were analysed in detail using Circular Dichroism (CD) and compared to their biochemical characteristics. Positive correlation (r = 0.74, p < 0.01) between the Tp and ratio of positive peak intensity to negative peak intensity (Rpn) in CD analysis suggested a higher thermal stability of ASC from warm-water fish, owing to a higher content of cyclic imino acids, such as proline and hydroxyproline (Hyp). Conversely, cold-water fish ASCs contain significantly higher levels of acyclic, hydroxyl groups carrying Ser. These results indicated that CD spectrum techniques including Rpn measurement are concise and helpful for direct detection of the triple helix structure of fish collagens, and that this structure is tightly linked to thermostability of this molecule.


Assuntos
Colágeno Tipo I/química , Hidroxiprolina/química , Prolina/química , Serina/química , Animais , Dicroísmo Circular , Peixes , Desnaturação Proteica , Temperatura
2.
Proc Natl Acad Sci U S A ; 117(15): 8503-8514, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32234784

RESUMO

The specific interaction of importins with nuclear localization signals (NLSs) of cargo proteins not only mediates nuclear import but also, prevents their aberrant phase separation and stress granule recruitment in the cytoplasm. The importin Transportin-1 (TNPO1) plays a key role in the (patho-)physiology of both processes. Here, we report that both TNPO1 and Transportin-3 (TNPO3) recognize two nonclassical NLSs within the cold-inducible RNA-binding protein (CIRBP). Our biophysical investigations show that TNPO1 recognizes an arginine-glycine(-glycine) (RG/RGG)-rich region, whereas TNPO3 recognizes a region rich in arginine-serine-tyrosine (RSY) residues. These interactions regulate nuclear localization, phase separation, and stress granule recruitment of CIRBP in cells. The presence of both RG/RGG and RSY regions in numerous other RNA-binding proteins suggests that the interaction of TNPO1 and TNPO3 with these nonclassical NLSs may regulate the formation of membraneless organelles and subcellular localization of numerous proteins.


Assuntos
Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Fragmentos de Peptídeos/metabolismo , Proteínas de Ligação a RNA/metabolismo , beta Carioferinas/metabolismo , Transporte Ativo do Núcleo Celular , Arginina/química , Arginina/metabolismo , Citoplasma/metabolismo , Glicina/química , Glicina/metabolismo , Células HeLa , Humanos , Fragmentos de Peptídeos/química , Ligação Proteica , Conformação Proteica , Proteínas de Ligação a RNA/química , Serina/química , Serina/metabolismo , Tirosina/química , Tirosina/metabolismo , beta Carioferinas/química
5.
J Med Chem ; 63(9): 4811-4823, 2020 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-32239932

RESUMO

PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. Synthetic antidiabetic drugs activating PPARγ are accompanied by serious undesirable side effects related to their agonism. In the search for new PPARγ regulators, inhibitors of PPARγ phosphorylation on S245 mediated by CDK5 represent an opportunity for the development of an improved generation of antidiabetic drugs acting through this nuclear receptor. We have employed a multidisciplinary approach, including protein-protein docking, X-ray crystallography, NMR, HDX, MD simulations, and site-directed mutagenesis to investigate conformational changes in PPARγ that impair the ability of CDK5 to interact with PPARγ and hence inhibit PPARγ phosphorylation. Finally, we describe an alternative inhibition mechanism adopted by a ligand bound far from the phosphorylation site.


Assuntos
PPAR gama/metabolismo , Fosforilação/efeitos dos fármacos , Sequência de Aminoácidos , Compostos de Bifenilo/química , Compostos de Bifenilo/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Proteínas do Tecido Nervoso/metabolismo , PPAR gama/antagonistas & inibidores , PPAR gama/química , PPAR gama/genética , Fenilpropionatos/química , Fenilpropionatos/metabolismo , Ligação Proteica , Conformação Proteica , Serina/química
6.
Biochemistry ; 59(13): 1309-1313, 2020 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-32207972

RESUMO

In a radical departure from the classical E1-E2-E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of Legionella pneumophila effectors utilize NAD+ to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD+ analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and the discovery and characterization of putative enzymes similar to members of the SidE family in other organisms. We also show their utility in studying enzymes that can reverse and inhibit this post-translational modification.


Assuntos
Proteínas de Bactérias/metabolismo , Bioquímica/métodos , Corantes Fluorescentes/química , Legionella pneumophila/metabolismo , Serina/metabolismo , Difosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Proteínas de Bactérias/química , Corantes Fluorescentes/metabolismo , Legionella pneumophila/química , Legionella pneumophila/genética , NAD/química , NAD/metabolismo , Serina/química , Ubiquitinação
7.
Food Chem ; 317: 126430, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32092612

RESUMO

Electrochemical Synchronous detection of cadmium (Cd(II)) and lead (Pb(II)) was obtained by acid treated multiwalled carbon nanotube (A-MWCNT) functionalized with hyaluronic acid (Hyalu) and this mixture was separately further modified with l-cysteine (l-Cys) and l-serine (l-Ser). Under the optimized circumstance best voltammetric responses were produced by A-MWCNT/Hyalu/l-Cys and A-MWCNT/Hyalu/l-Ser modified electrodes. The peak current was linearly dependent on the Cd(II) and Pb(II) concentrations in the range from 0.4 to 4 µg L-1. The sensitivities were calculated as 0.7 µA/nM (Cd(II)) and 3.5 µA/nM (Pb(II)) for A-MWCNT/Hyalu/l-Cys/GCE and 0.6 µA/nM (Cd(II)) and 2.6 µA/nM (Pb(II)) for A-MWCNT/Hyalu/l-Ser/GCE. From the calibration plot LODs were calculated to be 0.032 µg L-1 (Cd(II)) and 0.015 µg L-1 (Pb(II)) for A-MWCNT/Hyalu/l-Cys/GCE and 0.057 µg L-1 (Cd(II)) and 0.034 µg L-1 (Pb(II)) for A-MWCNT/Hyalu/l-Ser/GCE. Moreover, the proposed electrodes were subjected to the real sample application in honey, cocos nucifera and egg white.


Assuntos
Cádmio/análise , Análise de Alimentos/métodos , Contaminação de Alimentos/análise , Chumbo/análise , Nanocompostos/química , Cádmio/química , Calibragem , Cocos/química , Cisteína/química , Clara de Ovo/análise , Eletrodos , Análise de Alimentos/instrumentação , Mel/análise , Ácido Hialurônico/química , Chumbo/química , Limite de Detecção , Nanotubos de Carbono/química , Sensibilidade e Especificidade , Serina/química
8.
Macromol Rapid Commun ; 41(6): e1900583, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32009279

RESUMO

A unique cuboid spider silk from the outer egg sac of Nephila pilipes, with an unusual square cross-section, is disclosed. The structure-function relationships within this silk are first studied through structural characterization, mechanical measurement, protein conformation, and polypeptide signature of silk proteins. This silk maintains the higher stiffness property of egg sac silks, and also shows a species difference. Environmental response of the mechanical properties within this silk are observed. Synchrotron FTIR microspectroscopy is used to monitor the silk protein conformation in a single natural silk. The ß-sheet structure aligns parallel to the fiber axis with a content of 22% ± 2.6%. The de novo resulting polypeptide from the solid silk fibers are novel, and an abundant polar amino acid insertion is observed. Short polyalanine (An , n ≤ 3), alternating serine and alanine (S/A)X, and alternating glycine and alanine (G/A)X, GGX, and SSX dominates in the resulting de novo polypeptide. This accords with the composition pattern of other egg sac silk proteins, besides the rarely observed GGX. This study broadens the library of egg sac spider silks and provides a new perspective to uncover structure-function relationships in spider silk.


Assuntos
Aminoácidos/química , Fibroínas/química , Peptídeos/química , Seda/química , Alanina/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Fibroínas/ultraestrutura , Glicina/química , Teste de Materiais , Conformação Proteica em Folha beta , Serina/química , Seda/ultraestrutura , Aranhas/química , Relação Estrutura-Atividade
9.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 2): 65-73, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-32039887

RESUMO

Serine racemase (SR) is a pyridoxal 5'-phosphate (PLP)-containing enzyme that converts L-serine to D-serine, an endogenous co-agonist for the N-methyl-D-aspartate receptor (NMDAR) subtype of glutamate ion channels. SR regulates D-serine levels by the reversible racemization of L-serine to D-serine, as well as the catabolism of serine by α,ß-elimination to produce pyruvate. The modulation of SR activity is therefore an attractive therapeutic approach to disorders associated with abnormal glutamatergic signalling since it allows an indirect modulation of NMDAR function. In the present study, a 1.89 Šresolution crystal structure of the human SR holoenzyme (including the PLP cofactor) with four subunits in the asymmetric unit is described. Comparison of this new structure with the crystal structure of human SR with malonate (PDB entry 3l6b) shows an interdomain cleft that is open in the holo structure but which disappears when the inhibitor malonate binds and is enclosed. This is owing to a shift of the small domain (residues 78-155) in human SR similar to that previously described for the rat enzyme. This domain movement is accompanied by changes within the twist of the central four-stranded ß-sheet of the small domain, including changes in the φ-ψ angles of all three residues in the C-terminal ß-strand (residues 149-151). In the malonate-bound structure, Ser84 (a catalytic residue) points its side chain at the malonate and is preceded by a six-residue ß-strand (residues 78-83), but in the holoenzyme the ß-strand is only four residues (78-81) and His82 has φ-ψ values in the α-helical region of the Ramachandran plot. These data therefore represent a crystallographic platform that enables the structure-guided design of small-molecule modulators for this important but to date undrugged target.


Assuntos
Conformação Proteica , Racemases e Epimerases/química , Serina/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Domínios Proteicos
10.
Chem Commun (Camb) ; 56(10): 1537-1540, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-31922154

RESUMO

Although the underlying cause of Alzheimer's disease (AD) is not known, the extracellular deposition of ß-amyloid (Aß) is considered as a hallmark of AD brains. Evidence has shown the occurrence of d-Asp, isoAsp, and d-Ser residues in Aß, which may be indicative of and/or contribute to the neurodegeneration in AD patients. Herein, we have developed the first high-throughput profiling technique for all 20 isobaric Aß peptide epimers containing Asp, isoAsp, and Ser isomers using high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). This new analytical strategy allows the direct detection and identification of all possible Asp, isoAsp, and Ser stereoisomers in Aß, and may contribute to a better understanding of the pathogenesis of AD.


Assuntos
Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/análise , Doença de Alzheimer/metabolismo , Sequência de Aminoácidos , Peptídeos beta-Amiloides/química , Ácido Aspártico/química , Cromatografia Líquida de Alta Pressão , Humanos , Serina/química , Estereoisomerismo , Espectrometria de Massas em Tandem
11.
Chem Commun (Camb) ; 56(1): 74-77, 2020 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-31790117

RESUMO

We developed a new method for the de novo formation of fluorophores based on citrate (DNFC) in biological samples. Use of an amide coupling reagent and microwave irradiation greatly facilitates the fluorophore formation on peptides and proteins with N-terminal cysteine or serine. Since N-terminal cysteine and serine can form thiazolopyridone- or oxazolopyridone-based fluorophores emitting blue and green fluorescence, respectively, by the DNFC staining, each organelle, cell and tissue exhibited a characteristic fluorescence distribution. The DNFC staining is able to provide a new potential protocol for future cell imaging, histology and diagnosis.


Assuntos
Corantes Fluorescentes/metabolismo , Sondas Moleculares/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Animais , Linhagem Celular Tumoral , Ácido Cítrico/metabolismo , Cisteína/química , Fluorescência , Corantes Fluorescentes/química , Células HEK293 , Humanos , Camundongos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Sondas Moleculares/química , Células NIH 3T3 , Peptídeos/química , Estudo de Prova de Conceito , Proteínas/química , Piridonas/química , Piridonas/metabolismo , Serina/química , Tiazóis/química , Tiazóis/metabolismo
12.
J Biol Chem ; 295(5): 1402-1410, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31862734

RESUMO

ß-N-methylamino-l-alanine (BMAA) is a nonproteinogenic amino acid that has been associated with neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). BMAA has been found in human protein extracts; however, the mechanism by which it enters the proteome is still unclear. It has been suggested that BMAA is misincorporated at serine codons during protein synthesis, but direct evidence of its cotranslational incorporation is currently lacking. Here, using LC-MS-purified BMAA and several biochemical assays, we sought to determine whether any aminoacyl-tRNA synthetase (aaRS) utilizes BMAA as a substrate for aminoacylation. Despite BMAA's previously predicted misincorporation at serine codons, following a screen for amino acid activation in ATP/PPi exchange assays, we observed that BMAA is not a substrate for human seryl-tRNA synthetase (SerRS). Instead, we observed that BMAA is a substrate for human alanyl-tRNA synthetase (AlaRS) and can form BMAA-tRNAAla by escaping from the intrinsic AlaRS proofreading activity. Furthermore, we found that BMAA inhibits both the cognate amino acid activation and the editing functions of AlaRS. Our results reveal that, in addition to being misincorporated during translation, BMAA may be able to disrupt the integrity of protein synthesis through multiple different mechanisms.


Assuntos
Alanina-tRNA Ligase/metabolismo , Diamino Aminoácidos/metabolismo , Aminoacilação de RNA de Transferência , Alanina/química , Alanina/metabolismo , Diamino Aminoácidos/química , Cromatografia Líquida , Expressão Gênica , Humanos , Cinética , Espectrometria de Massas , Serina/química , Serina/metabolismo , Serina-tRNA Ligase/metabolismo
13.
J Phys Chem Lett ; 10(24): 7872-7877, 2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31790267

RESUMO

The deposition of coassemblies made of the small presynaptic protein, α-synuclein, and lipids in the brains of patients is the hallmark of Parkinson's disease. In this study, we used natural abundance 13C and 31P magic-angle spinning nuclear magnetic resonance spectroscopy together with cryo-electron microscopy and differential scanning calorimetry to characterize the fibrils formed by α-synuclein in the presence of vesicles made of 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine or 1,2-dilauroyl-sn-glycero-3-phospho-L-serine. Our results show that these lipids coassemble with α-synuclein molecules to give thin and curly amyloid fibrils. The coassembly leads to slower and more isotropic reorientation of lipid molecular segments and a decrease in both the temperature and enthalpy of the lipid chain-melting compared with those in the protein-free lipid lamellar phase. These findings provide new insights into the properties of lipids within protein-lipid assemblies that can be associated with Parkinson's disease.


Assuntos
Amiloide/química , Bicamadas Lipídicas/química , alfa-Sinucleína/química , Cinética , Estrutura Molecular , Transição de Fase , Ligação Proteica , Serina/química , Relação Estrutura-Atividade , Termodinâmica , Temperatura de Transição
14.
PLoS One ; 14(11): e0225510, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31751425

RESUMO

To establish a strategy for identifying protein-N-myristoylation-dependent phosphorylation of cellular proteins, Phos-tag SDS-PAGE was performed on wild-type (WT) and nonmyristoylated mutant (G2A-mutant) FMNL2 and FMNL3, phosphorylated N-myristoylated model proteins expressed in HEK293 cells. The difference in the banding pattern in Phos-tag SDS-PAGE between the WT and G2A-mutant FMNL2 indicated the presence of N-myristoylation-dependent phosphorylation sites in FMNL2. Phos-tag SDS-PAGE of FMNL2 mutants in which the putative phosphorylation sites listed in PhosphoSitePlus (an online database of phosphorylation sites) were changed to Ala revealed that Ser-171 and Ser-1072 are N-myristoylation-dependent phosphorylation sites in FMNL2. Similar experiments with FMNL3 demonstrated that N-myristoylation-dependent phosphorylation occurs at a single Ser residue at position 174, which is a Ser residue conserved between FMNL2 and FMNL3, corresponding to Ser-171 in FMNL2. The facts that phosphorylation of Ser-1072 in FMNL2 has been shown to play a critical role in integrin ß1 internalization mediated by FMNL2 and that Ser-171 in FMNL2 and Ser-174 in FMNL3 are novel putative phosphorylation sites conserved between FMNL2 and FMNL3 indicate that the strategy used in this study is a useful tool for identifying and characterizing physiologically important phosphorylation reactions occurring on N-myristoylated proteins.


Assuntos
Forminas/metabolismo , Piridinas/química , Serina/química , Animais , Células COS , Chlorocebus aethiops , Eletroforese em Gel de Poliacrilamida , Forminas/química , Forminas/genética , Células HEK293 , Humanos , Mutação , Fosforilação
15.
Chirality ; 31(12): 1043-1052, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31667899

RESUMO

A novel chiral derivatization reagent, the N-[1-oxo-5-(triphenylphosphonium)pentyl]- (R)-1,3-thiazolidinyl-4-N-hydroxysuccinimide ester bromide salt (OTPTHE), was developed for the separation and selective detection of chiral DL-amino acids by RP-HPLC analysis. The OTPTHE reacted with DL-amino acids at 60°C maintained for 30 minutes in the presence of 100 mM borate buffer (pH 9.5). The separability of the diastereomeric derivatives was evaluated in terms of the resolution value (Rs) using 13 kinds of DL-amino acids, which were completely separated by reversed-phase chromatography using C18 column at 254 nm. The Rs of the DL-amino acids varied from 1.62 to 2.51. As for the application of the DL-amino acids, the determination of DL-Ser in the human plasma of healthy volunteers was performed based on our developed method. It was shown that linear calibrations were available with high coefficients of correlation (r2 > 0.9997). The limit of detection (S/N = 3) of the DL-Ser enantiomers was 5.0 pmol; the relative standard deviations of the intraday and interday variations were below 4.56%; the accuracy ranged between 95.40%-110.06% and 95.45%-109.80%, respectively; the mean recoveries (%) of the DL-Ser spiked in the human plasma were 99.49%-103.74%. The amounts of DL-Ser in the human plasma of healthy volunteers were determined.


Assuntos
Serina/sangue , Serina/química , Succinimidas/química , Tiazolidinas/química , Calibragem , Fracionamento Químico , Cromatografia Líquida , Humanos , Indicadores e Reagentes/química , Serina/isolamento & purificação , Estereoisomerismo
16.
J Korean Med Sci ; 34(42): e266, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31674157

RESUMO

BACKGROUND: Apart from its blood pressure-lowering effect by blocking the renin-angiotensin-aldosterone system, telmisartan, an angiotensin II type 1 receptor blocker (ARB), exhibits various ancillary effects including cardiovascular protective effects in vitro. Nonetheless, the protective effects of telmisartan in cerebrocardiovascular diseases are somewhat variable in large-scale clinical trials. Dysregulation of endothelial nitric oxide (NO) synthase (eNOS)-derived NO contributes to the developments of various vascular diseases. Nevertheless, the direct effects of telmisartan on endothelial functions including NO production and vessel relaxation, and its action mechanism have not been fully elucidated. Here, we investigated the mechanism by which telmisartan regulates NO production and vessel relaxation in vitro and in vivo. METHODS: We measured nitrite levels in culture medium and mouse serum, and performed inhibitor studies and western blot analyses using bovine aortic endothelial cells (BAECs) and a hyperglycemic mouse model. To assess vessel reactivity, we performed acetylcholine (ACh)-induced vessel relaxation assay on isolated rat aortas. RESULTS: Telmisartan decreased NO production in normoglycemic and hyperglycemic BAECs, which was accompanied by reduced phosphorylation of eNOS at Ser1179 (p-eNOS-Ser1179). Telmisartan increased the expression of protein phosphatase 2A catalytic subunit (PP2Ac) and co-treatment with okadaic acid completely restored telmisartan-inhibited NO production and p-eNOS-Ser1179 levels. Of the ARBs tested (including losartan and fimasartan), only telmisartan decreased NO production and p-eNOS-Ser1179 levels, and enhanced PP2Ac expression. Co-treatment with GW9662 had no effect on telmisartan-induced changes. In line with in vitro observations, telmisartan reduced serum nitrite and p-eNOS-Ser1179 levels, and increased PP2Ac expression in high fat diet-fed mice. Furthermore, telmisartan attenuated ACh-induced rat aorta relaxation. CONCLUSION: We demonstrated that telmisartan inhibited NO production and vessel relaxation at least in part by PP2A-mediated eNOS-Ser1179 dephosphorylation in a peroxisome proliferator-activated receptor γ-independent manner. These results may provide a mechanism that explains the inconsistent cerebrocardiovascular protective effects of telmisartan.


Assuntos
Óxido Nítrico Sintase Tipo III/química , Óxido Nítrico/metabolismo , Proteína Fosfatase 2/metabolismo , Telmisartan/farmacologia , Acetilcolina/química , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Aorta/metabolismo , Bovinos , Modelos Animais de Doenças , Endotélio Vascular/patologia , Hiperglicemia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nitritos/química , Fosforilação , Proteína Fosfatase 2C/metabolismo , Ratos , Serina/química
17.
J Agric Food Chem ; 67(46): 12953-12961, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31638789

RESUMO

Most common sphingolipids are comprised of "typical" sphingoid bases (sphinganine, sphingosine, and structurally related compounds) and are produced via the condensation of l-serine with a fatty acyl-CoA by serine palmitoyltransferase. Some organisms, including mammals, also produce "atypical" sphingoid bases that lack a 1-hydroxyl group as a result of the utilization of l-alanine or glycine instead of l-serine, resulting in the formation of 1-deoxy- or 1-desoxymethylsphingoid bases, respectively. Elevated production of "atypical" sphingolipids has been associated with human disease, but 1-deoxysphingoid bases have also been found to have potential as anticancer compounds, hence, the importance of knowing more about the occurrence of these compounds in food. Most of the "typical" and "atypical" sphingoid bases are found as the N-acyl metabolites (e.g., ceramides and 1-deoxyceramides) in mammals, but this has not been uniformly assessed in previous studies nor determined in consumed food. Therefore, we developed a method for the quantitative analysis of "typical" and "atypical" sphingoid bases and their N-acyl derivatives by reverse-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. On the basis of these analyses, there was considerable variability in the amounts and molecular subspecies of atypical sphingoid bases and their N-acyl metabolites found in different edible sources. These findings demonstrate that a broader assessment of the types of sphingolipids in foods is needed because some diets might contain sufficient amounts of atypical as well as typical sphingolipids that could have beneficial or possibly deleterious effects on human health.


Assuntos
Acil Coenzima A/química , Esfingolipídeos/química , Acil Coenzima A/metabolismo , Serina/química , Serina/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Esfingolipídeos/metabolismo
18.
Molecules ; 24(20)2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31623098

RESUMO

Oleoyl serine (OS), an endogenous fatty acyl amide (FAA) found in bone, has been shown to have an anti-osteoporotic effect. OS, being an amide, can be hydrolyzed in the body by amidases. Hindering its amide bond by introducing adjacent substituents has been demonstrated as a successful method for prolonging its skeletal activity. Here, we tested the therapeutic efficacy of two methylated OS derivatives, oleoyl α-methyl serine (HU-671) and 2-methyl-oleoyl serine (HU-681), in an ovariectomized mouse model for osteoporosis by utilizing combined micro-computed tomography, histomorphometry, and cell culture analyses. Our findings indicate that daily treatment for 6 weeks with OS or HU-671 completely rescues bone loss, whereas HU-681 has only a partial effect. The increased bone density was primarily due to enhanced trabecular thickness and number. Moreover, the most effective dose of HU-671 was 0.5 mg/kg/day, an order of magnitude lower than with OS. The reversal of bone loss resulted from increased bone formation and decreased bone resorption, as well as reversal of bone marrow adiposity. These results were further confirmed by determining the serum levels of osteocalcin and type 1 collagen C-terminal crosslinks, as well as demonstrating the enhanced antiadipogenic effect of HU-671. Taken together, these data suggest that methylation interferes with OS's metabolism, thus enhancing its effects by extending its availability to its target cells.


Assuntos
Adiposidade/efeitos dos fármacos , Medula Óssea/efeitos dos fármacos , Medula Óssea/patologia , Ácidos Oleicos/química , Osteoporose/etiologia , Osteoporose/metabolismo , Serina/análogos & derivados , Serina/farmacologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Medula Óssea/diagnóstico por imagem , Modelos Animais de Doenças , Feminino , Camundongos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoporose/diagnóstico , Ovariectomia/efeitos adversos , Serina/química , Microtomografia por Raio-X
19.
Int J Mol Sci ; 20(20)2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31658747

RESUMO

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder caused by the mutation of cholesterol-transporting proteins. In addition, early treatment is important for good prognosis of this disease because of the progressive neurodegeneration. However, the diagnosis of this disease is difficult due to a variety of clinical spectrum. Lysosphingomyelin-509, which is one of the most useful biomarkers for NPC, was applied for the rapid and easy detection of NPC. The fact that its chemical structure was unknown until recently implicates the unrevealed pathophysiology and molecular mechanisms of NPC. In this study, we aimed to elucidate the structure of lysosphingomyelin-509 by various mass spectrometric techniques. As our identification strategy, we adopted analytical and organic chemistry approaches to the serum of patients with NPC. Chemical derivatization and hydrogen abstraction dissociation-tandem mass spectrometry were used for the determination of function groups and partial structure, respectively. As a result, we revealed the exact structure of lysosphingomyelin-509 as N-acylated and O-phosphocholine adducted serine. Additionally, we found that a group of metabolites with N-acyl groups were increased considerably in the serum/plasma of patients with NPC as compared to that of other groups using targeted lipidomics analysis. Our techniques were useful for the identification of lysosphingomyelin-509.


Assuntos
Lipídeos/química , Lipídeos/isolamento & purificação , Doença de Niemann-Pick Tipo C/diagnóstico , Fosforilcolina/química , Fosforilcolina/isolamento & purificação , Serina/química , Biomarcadores/sangue , Feminino , Humanos , Masculino , Doença de Niemann-Pick Tipo C/metabolismo , Fosforilcolina/metabolismo , Serina/metabolismo , Espectrometria de Massas em Tandem/métodos
20.
Int J Pharm ; 570: 118653, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31472218

RESUMO

Co-amorphous mixtures have been demonstrated to represent a promising approach for enhancing the dissolution of poorly water-soluble drugs. However, little is known of their permeability properties, especially through biological membranes, or about the relationship between their dissolution and permeability. In the present study, co-amorphous glibenclamide (GBC) mixtures with two amino acids, arginine (ARG) and serine (SER), in molar ratios of 1:1 were prepared by cryomilling. Their dissolution and permeability properties were studied in side-by-side diffusion chambers using cell layers containing Madine Darby kidney cells overexpressing P-glycoprotein (Pgp) transporters (MDCKII-MDR1), as Pgp may influence the absorption of GBC. Furthermore, two other compounds, the flavonoid quercetin (QRT) which is a Pgp inhibitor and the surfactant, sodium lauryl sulfate (SLS), were used as excipients to investigate if they improved either passive or active diffusion of GBC. In addition, amorphous QRT and a co-amorphous mixture of GBC and QRT (1:1) were characterized with respect to their solid-state properties and physical stability. It was demonstrated that co-amorphous GBC mixtures exhibited superior dissolution properties over the corresponding physical mixtures and amorphous GBC. Furthermore, the co-amorphous GBC-ARG-SLS mixture exhibited a 9-fold increase in permeating through the MDCKII-MDR1 cell layer as compared to the corresponding physical mixture. There was a correlation between the dissolution and permeability area under curve (AUC) values, evidence that the main mechanism behind the improved permeability of co-amorphous mixtures was their improved dissolution. The simultaneous dissolution/permeation testing with side-by-side diffusion chambers and MDCKII-MDR1 cells proved to be a feasible method for evaluating the dissolution/permeation interplay of amorphous compounds.


Assuntos
Glibureto/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/química , Animais , Arginina/química , Cães , Composição de Medicamentos/métodos , Flavonoides/química , Células Madin Darby de Rim Canino , Permeabilidade/efeitos dos fármacos , Difração de Pó/métodos , Quercetina/química , Serina/química , Solubilidade/efeitos dos fármacos
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