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1.
Mol Cell ; 81(22): 4622-4634.e8, 2021 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-34551282

RESUMO

AKT is a serine/threonine kinase that plays an important role in metabolism, cell growth, and cytoskeletal dynamics. AKT is activated by two kinases, PDK1 and mTORC2. Although the regulation of PDK1 is well understood, the mechanism that controls mTORC2 is unknown. Here, by investigating insulin receptor signaling in human cells and biochemical reconstitution, we found that insulin induces the activation of mTORC2 toward AKT by assembling a supercomplex with KRAS4B and RHOA GTPases, termed KARATE (KRAS4B-RHOA-mTORC2 Ensemble). Insulin-induced KARATE assembly is controlled via phosphorylation of GTP-bound KRAS4B at S181 and GDP-bound RHOA at S188 by protein kinase A. By developing a KARATE inhibitor, we demonstrate that KRAS4B-RHOA interaction drives KARATE formation. In adipocytes, KARATE controls insulin-dependent translocation of the glucose transporter GLUT4 to the plasma membrane for glucose uptake. Thus, our work reveals a fundamental mechanism that activates mTORC2 toward AKT in insulin-regulated glucose homeostasis.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/química , Glucose/metabolismo , Insulina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/química , Proteína rhoA de Ligação ao GTP/química , Células 3T3-L1 , Adipócitos/citologia , Animais , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Dictyostelium , Transportador de Glucose Tipo 4/metabolismo , Guanosina Difosfato/química , Guanosina Trifosfato/química , Células HEK293 , Humanos , Camundongos , Fosforilação , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas p21(ras)/química , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
2.
Mol Cell ; 80(4): 621-632.e6, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33152269

RESUMO

Mitochondria are highly dynamic organelles that continuously grow, divide, and fuse. The division of mitochondria is crucial for human health. During mitochondrial division, the mechano-guanosine triphosphatase (GTPase) dynamin-related protein (Drp1) severs mitochondria at endoplasmic reticulum (ER)-mitochondria contact sites, where peripheral ER tubules interact with mitochondria. Here, we report that Drp1 directly shapes peripheral ER tubules in human and mouse cells. This ER-shaping activity is independent of GTP hydrolysis and located in a highly conserved peptide of 18 amino acids (termed D-octadecapeptide), which is predicted to form an amphipathic α helix. Synthetic D-octadecapeptide tubulates liposomes in vitro and the ER in cells. ER tubules formed by Drp1 promote mitochondrial division by facilitating ER-mitochondria interactions. Thus, Drp1 functions as a two-in-one protein during mitochondrial division, with ER tubulation and mechano-GTPase activities.


Assuntos
Dinaminas/metabolismo , Dinaminas/fisiologia , Retículo Endoplasmático/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Trifosfato/metabolismo , Mitocôndrias/metabolismo , Animais , Dinaminas/genética , Retículo Endoplasmático/efeitos dos fármacos , GTP Fosfo-Hidrolases/genética , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Dinâmica Mitocondrial , Oligopeptídeos/farmacologia
3.
J Am Chem Soc ; 146(32): 22193-22207, 2024 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-38963258

RESUMO

Glycans cover the cell surface to form the glycocalyx, which governs a myriad of biological phenomena. However, understanding and regulating glycan functions is extremely challenging due to the large number of heterogeneous glycans that engage in intricate interaction networks with diverse biomolecules. Glycocalyx-editing techniques offer potent tools to probe their functions. In this study, we devised a HaloTag-based technique for glycan manipulation, which enables the introduction of chemically synthesized glycans onto a specific protein (protein of interest, POI) and concurrently incorporates fluorescent units to attach homogeneous, well-defined glycans to the fluorescence-labeled POIs. Leveraging this HaloTag-based glycan-display system, we investigated the influence of the interactions between Gal-3 and various N-glycans on protein dynamics. Our analyses revealed that glycosylation modulates the lateral diffusion of the membrane proteins in a structure-dependent manner through interaction with Gal-3, particularly in the context of the Gal-3-induced formation of the glycan network (galectin lattice). Furthermore, N-glycan attachment was also revealed to have a significant impact on the extracellular vesicle-loading of membrane proteins. Notably, our POI-specific glycan introduction does not disrupt intact glycan structures, thereby enabling a functional analysis of glycans in the presence of native glycan networks. This approach complements conventional glycan-editing methods and provides a means for uncovering the molecular underpinnings of glycan functions on the cell surface.


Assuntos
Vesículas Extracelulares , Galectinas , Proteínas de Membrana , Polissacarídeos , Polissacarídeos/química , Polissacarídeos/metabolismo , Glicosilação , Galectinas/metabolismo , Galectinas/química , Vesículas Extracelulares/química , Vesículas Extracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química , Humanos , Difusão , Membrana Celular/metabolismo , Membrana Celular/química
4.
EMBO J ; 39(24): e105074, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33200421

RESUMO

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin-related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin-related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1-knockout cells. Re-activation of Opa1 and Mitofusin 1 in Drp1-knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.


Assuntos
Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Mitocôndrias/metabolismo , Estresse Fisiológico/fisiologia , Animais , Dinaminas/genética , Dinaminas/metabolismo , Metabolismo Energético , GTP Fosfo-Hidrolases/metabolismo , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Metaloproteases/metabolismo , Camundongos , Mitocôndrias/genética , Dinâmica Mitocondrial/fisiologia , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Estresse Fisiológico/genética , Transcriptoma
5.
Chemistry ; 29(3): e202202387, 2023 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-36254793

RESUMO

Type-1 iodothyronine deiodinase (ID-1) catalyzes the reductive elimination of 5'-I and 5-I on the phenolic and tyrosyl rings of thyroxine (T4), respectively. Chemically verifying whether I atoms with different chemical properties undergo deiodination through a common mechanism is challenging. Herein, we report the modeling of ID-1 using aliphatic diselenide (Se-Se) and selenenylsulfide (Se-S) compounds. Mechanistic investigations of deiodination using the ID-1-like reagents suggested that the 5'-I and 5-I deiodinations proceed via the same mechanism through an unstable intermediate containing a Se⋅⋅⋅I halogen bond between a selenolate anion, reductively produced from Se-Se (or Se-S) in the compound, and an I atom in T4. Moreover, imidazolium and thiol groups, which may act as general acid catalysts, promoted the heterolytic cleavage of the C-I bond in the Se⋅⋅⋅I intermediate, which is the rate-determining step, by donating a proton to the C atom.


Assuntos
Iodeto Peroxidase , Tiroxina , Iodeto Peroxidase/química , Tiroxina/química , Halogênios/química , Catálise , Fenóis , Tri-Iodotironina/química
6.
Chembiochem ; 23(5): e202100394, 2022 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-34350692

RESUMO

This study developed dipeptide-conjugated 1,2-diselenan-4-amine (1), i. e., 1-Xaa-His, as a new class of S-denitrosylase mimic. The synthesized compounds, especially 1-Pro-His, remarkably promoted S-denitrosylation of nitrosothiols (RSNO) via a catalytic cycle involving the reversible redox reaction between the diselenide and its corresponding diselenol ([SeH,SeH]) form with coexisting reductant thiols (R'SH), during which the [SeH,SeH] form as a key reactive species reduces RSNO to the corresponding thiol (RSH). Structural analyses of 1-Pro-His suggested that the peptide backbone of [SeH,SeH] is rigidly bent to form a γ-turn, possibly including an NH⋅⋅⋅Se hydrogen bond between the imidazole ring of His and selenol group, thus stabilizing the [SeH,SeH] form thermodynamically, and dramatically enhancing the catalytic activity. Furthermore, the synthetic compounds were found to prohibit S-nitrosylation-induced protein misfolding in the presence of RSNO, eventually implying their potential as a drug seed for misfolding diseases caused by the dysregulation of the S-denitrosylation system.


Assuntos
Dipeptídeos , Prolina , Oxirredução , Proteínas , Compostos de Sulfidrila
7.
FASEB J ; 34(3): 3838-3854, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31970839

RESUMO

The tumor microenvironment (TME) formation involving host cells and cancer cells through cell adhesion molecules (CAMs) is essential for the multiple steps of cancer metastasis and growth. Sphingomyelin synthase 2 (SMS2) is involved in inflammatory diseases such as obesity and diabetes mellitus by regulation of the SM/ceramide balance. However, the involvement of SMS2 in TME formation and metastasis is largely unknown. Here, we report that SMS2-deficient (SMS2-KO) mice show suppressed the EL4 cell infiltration to liver and prolonged survival time. ICAM-1 was identified as a candidate for the inhibition of TME formation in immortalized mouse embryonic fibroblasts (tMEFs) from mRNA array analysis for CAMs. Reduced SM/ceramide balance in SMS2-KO tMEFs suppressed the attachment of EL4 cells through transcriptional reduction of ICAM-1 by the inhibition of NF-κB activation. TNF-α-induced NF-κB activation and subsequent induction of ICAM-1 were suppressed in SMS2-KO tMEFs but restored by SMS2 re-introduction. In the EL4 cell infiltration mouse model, EL4 injection increased ICAM-1 expression in WT liver but not in SMS2-KO mouse liver. Therefore, inhibition of SMS2 may be a therapeutic target to suppress the infiltration of malignant lymphoma.


Assuntos
Molécula 1 de Adesão Intercelular/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida , Modelos Animais de Doenças , Citometria de Fluxo , Glucosiltransferases/metabolismo , Imuno-Histoquímica , Molécula 1 de Adesão Intercelular/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Knockout , Camundongos Mutantes , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espectrometria de Massas em Tandem , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Fator de Necrose Tumoral alfa/farmacologia
8.
Bioorg Med Chem ; 29: 115866, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33203607

RESUMO

Amphiphilic derivatives of (±)-trans-1,2-diselenane-4,5-diol (DSTox) decorated with long alkyl chains or aromatic substituents via ester linkages were applied as glutathione peroxidase (GPx)-like catalysts. The reduction of H2O2 with the diselenide catalysts was accelerated through a GPx-like catalytic cycle, in which the diselenide (Se-Se) bond was reduced to the diselenolate form ([Se-,Se-]) by coexisting dithiothreitol, and the generated highly active [Se-,Se-] subsequently reduced H2O2 to H2O retrieving the original Se-Se form. In the lipid peroxidation of lecithin/cholesterol liposomes induced by 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), on the other hand, the Se-Se form directly reduced lipid peroxide (LOOH) to the corresponding alcohol (LOH), inhibiting the radical chain reaction, to exert the antioxidative effect. Thus, the two GPx-like catalytic cycles can be switched depending on the peroxide substrates. Furthermore, hydrophilic compounds with no or short alkyl groups (C3) showed high antioxidative activities for the catalytic reduction of H2O2, while lipophilic compounds with long alkyl chains (C6-C14) or aromatic substituents were more effective antioxidants against lipid peroxidation. In addition, these compounds showed low cytotoxicity in cultured HeLa cells and exhibited sufficient anti-lipid peroxidative activities, suggesting their potentials as selenium-based antioxidative drugs.


Assuntos
Antioxidantes/química , Peróxidos/química , Tensoativos/química , Antioxidantes/farmacologia , Catálise , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HeLa , Humanos , Peróxido de Hidrogênio/química , Estrutura Molecular , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Estereoisomerismo , Relação Estrutura-Atividade , Tensoativos/farmacologia , Células Tumorais Cultivadas
9.
Molecules ; 26(1)2021 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-33401729

RESUMO

In the last few decades, development of novel experimental techniques, such as new types of disulfide (SS)-forming reagents and genetic and chemical technologies for synthesizing designed artificial proteins, is opening a new realm of the oxidative folding study where peptides and proteins can be folded under physiologically more relevant conditions. In this review, after a brief overview of the historical and physicochemical background of oxidative protein folding study, recently revealed folding pathways of several representative peptides and proteins are summarized, including those having two, three, or four SS bonds in the native state, as well as those with odd Cys residues or consisting of two peptide chains. Comparison of the updated pathways with those reported in the early years has revealed the flexible nature of the protein folding pathways. The significantly different pathways characterized for hen-egg white lysozyme and bovine milk α-lactalbumin, which belong to the same protein superfamily, suggest that the information of protein folding pathways, not only the native folded structure, is encoded in the amino acid sequence. The application of the flexible pathways of peptides and proteins to the engineering of folded three-dimensional structures is an interesting and important issue in the new realm of the current oxidative protein folding study.


Assuntos
Dissulfetos/química , Peptídeos/química , Peptídeos/metabolismo , Proteínas/química , Proteínas/metabolismo , Animais , Bovinos , Cisteína/química , Lactalbumina/química , Lactalbumina/metabolismo , Muramidase/química , Muramidase/metabolismo , Oxirredução , Conformação Proteica , Dobramento de Proteína
10.
Biochem Biophys Res Commun ; 532(1): 19-24, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-32826055

RESUMO

Glycolipid metabolism occurs in the Golgi apparatus, but the detailed mechanisms have not yet been elucidated. We used fluorescently labeled glycolipids to analyze glycolipid composition and localization changes and shed light on glycolipid metabolism. In a previous study, the fatty chain of lactosyl ceramide was fluorescently labeled with BODIPY (LacCer-BODIPY) before being introduced into cultured cells to analyze the cell membrane glycolipid recycling process. However, imaging analysis of glycolipid recycling is difficult because of limited spatial resolution. Therefore, we examined the microscopic conditions that allow the temporal analysis of LacCer-BODIPY trafficking and localization. We observed that the glycolipid fluorescent probe migrated from the cell membrane to intracellular organelles before returning to the cell membrane. We used confocal microscopy to observe co-localization of the glycolipid probe with endosomes and Golgi markers, demonstrating that it recycles mainly through the trans-Golgi network (TGN). Here, a glycolipid recycling pathway was observed that did not require the lipids to pass through the lysosome.


Assuntos
Glicolipídeos/metabolismo , Animais , Transporte Biológico Ativo , Compostos de Boro , Células CHO , Membrana Celular/metabolismo , Cricetulus , Endossomos/metabolismo , Corantes Fluorescentes , Complexo de Golgi/metabolismo , Lactosilceramidas , Lisossomos/metabolismo , Microscopia Confocal , Modelos Biológicos , Análise Espaço-Temporal , Imagem com Lapso de Tempo , Rede trans-Golgi/metabolismo
11.
Org Biomol Chem ; 18(19): 3724-3733, 2020 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-32364197

RESUMO

Glycosphingolipids (GSLs) are a group of molecules composed of a hydrophilic glycan part and a hydrophobic ceramide creating a diverse family. GSLs are de novo synthesised from ceramides at the endoplasmic reticulum and Golgi apparatus, and transported to the outer surface of the plasma membrane. It has been known that the glycan structures of GSLs change reflecting disease states. We envisioned that analysing the glycan pattern of GSLs enables distinguishing diseases. For this purpose, we utilised a fluorescently tagged compound, LacCerBODIPY (1). At first, compound 1 was taken up by cultured PC12D cells and transformed into various GSLs. As a result, changes in the GSL patterns of differentiation states of the cells were successfully observed by using an analysis platform, nano-liquid chromatography (LC)-fluorescence detection (FLD)-electrospray ionisation (ESI)-mass spectrometry (MS), which could quantify and provide molecular ions simultaneously. We found that compound 1 remained for about 10 min on the plasma membrane before it was converted into other GSLs. We therefore investigated a more rapid way to discriminate different cellular states by fluorescence recovery after photobleaching, which revealed that it is possible to distinguish the differentiation states as well.


Assuntos
Compostos de Boro/metabolismo , Membrana Celular/metabolismo , Lactosilceramidas/metabolismo , Polissacarídeos/metabolismo , Animais , Compostos de Boro/química , Membrana Celular/química , Lactosilceramidas/química , Estrutura Molecular , Células PC12 , Polissacarídeos/química , Ratos
12.
Chemistry ; 25(55): 12751-12760, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31390113

RESUMO

At the redox-active center of thioredoxin reductase (TrxR), a selenenyl sulfide (Se-S) bond is formed between Cys497 and Sec498, which is activated into the thiolselenolate state ([SH,Se- ]) by reacting with a nearby dithiol motif ([SHCys59 ,SHCys64 ]) present in the other subunit. This process is achieved through two reversible steps: an attack of a cysteinyl thiol of Cys59 at the Se atom of the Se-S bond and a subsequent attack of a remaining thiol at the S atom of the generated mixed Se-S intermediate. However, it is not clear how the kinetically unfavorable second step progresses smoothly in the catalytic cycle. A model study that used synthetic selenenyl sulfides, which mimic the active site structure of human TrxR comprising Cys497, Sec498, and His472, suggested that His472 can play a key role by forming a hydrogen bond with the Se atom of the mixed Se-S intermediate to facilitate the second step. In addition, the selenenyl sulfides exhibited a defensive ability against H2 O2 -induced oxidative stress in cultured cells, which suggests the possibility for medicinal applications to control the redox balance in cells.

13.
J Lipid Res ; 59(11): 2181-2187, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30242108

RESUMO

Intestinal cholesterol absorption is a key regulator of systemic cholesterol homeostasis. Excessive dietary cholesterol and its intestinal uptake lead to hypercholesterolemia, a major risk factor for cardiovascular disease. Intestinal cholesterol uptake is mediated by Niemann-Pick C1-like 1 (NPC1L1), a transmembrane protein localized in membrane microdomains (lipid rafts) enriched in gangliosides and cholesterol. The roles of gangliosides, such as monosialodihexosylganglioside (GM3) and its synthesizing enzyme GM3 synthase (GM3S), in NPC1L1-dependent cholesterol uptake have not been examined previously. Here, we examined NPC1L1-dependent cholesterol uptake in a cell model as well as in wild-type and apoE-deficient mice fed normal or high-cholesterol diets. We showed that NPC1L1-dependent cholesterol uptake was impaired in GM3S-deficient cells and that GM3S deficiency promoted resistance to hypercholesterolemia in both wild-type and apoE-deficient mice fed the high-cholesterol but not the normal diet. Our findings suggest that GM3 and related gangliosides are essential for NPC1L1-mediated intestinal cholesterol absorption and are potential targets for hypercholesterolemia therapy.


Assuntos
Colesterol/sangue , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Transporte Biológico , Gangliosídeo G(M3) , Células HEK293 , Humanos , Hipercolesterolemia/metabolismo , Imuno-Histoquímica , Absorção Intestinal , Lipoproteínas/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espectrometria de Massas em Tandem
14.
Chembiochem ; 19(3): 207-211, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29197144

RESUMO

The protein disulfide isomerase (PDI) family, found in the endoplasmic reticulum (ER) of the eukaryotic cell, catalyzes the formation and cleavage of disulfide bonds and thereby helps in protein folding. A decrease in PDI activity under ER stress conditions leads to protein misfolding, which is responsible for the progression of various human diseases, such as Alzheimer's, Parkinson's, diabetes mellitus, and atherosclerosis. Here we report that water-soluble cyclic diselenides mimic the multifunctional activity of the PDI family by facilitating oxidative folding, disulfide formation/reduction, and repair of the scrambled disulfide bonds in misfolded proteins.


Assuntos
Compostos Organosselênicos/metabolismo , Oxirredutases/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína , Biocatálise , Sobrevivência Celular , Dissulfetos/química , Dissulfetos/metabolismo , Retículo Endoplasmático/enzimologia , Células Eucarióticas/enzimologia , Células HEK293 , Humanos , Estrutura Molecular , Compostos Organosselênicos/química , Oxirredutases/química , Isomerases de Dissulfetos de Proteínas/química , Solubilidade , Água/química
15.
Proc Natl Acad Sci U S A ; 112(46): E6388-96, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26540727

RESUMO

Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms.


Assuntos
Bryopsida , Sistema de Sinalização das MAP Quinases/fisiologia , Pressão Osmótica/fisiologia , Proteínas de Plantas , Quinases raf , Sequência de Aminoácidos , Bryopsida/enzimologia , Bryopsida/genética , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Quinases raf/genética , Quinases raf/metabolismo
16.
Biochemistry ; 56(42): 5644-5653, 2017 10 24.
Artigo em Inglês | MEDLINE | ID: mdl-29022711

RESUMO

Selenoglutathione (GSeH) is a selenium analogue of naturally abundant glutathione (GSH). In this study, this water-soluble small tripeptide was synthesized in a high yield (up to 98%) as an oxidized diselenide form, i.e., GSeSeG (1), by liquid-phase peptide synthesis (LPPS). Obtained 1 was applied to the investigation of the glutathione peroxidase (GPx)-like catalytic cycle. The important intermediates, i.e., GSe- and GSeSG, besides GSeO2H were characterized by 77Se NMR spectroscopy. Thiol exchange of GSeSG with various thiols, such as cysteine and dithiothreitol, was found to promote the conversion to GSe- significantly. In addition, disproportionation of GSeSR to 1 and RSSR, which would be initiated by heterolytic cleavage of the Se-S bond and catalyzed by the generated selenolate, was observed. On the basis of these redox behaviors, it was proposed that the heterolytic cleavage of the Se-S bond can be facilitated by the interaction between the Se atom and an amino or aromatic group, which is present at the GPx active site. On the other hand, when a catalytic amount of 1 was reacted with scrambled 4S species of RNase A in the presence of NADPH and glutathione reductase, native protein was efficiently regenerated, suggesting a potential use of 1 to repair misfolded proteins through reduction of the non-native SS bonds.


Assuntos
Dissulfetos/química , Glutationa Peroxidase/química , Glutationa/análogos & derivados , Glutationa/química , Ribonuclease Pancreático/química , Selênio/química , Glutationa/síntese química , Oxirredução
17.
Metrologia ; 54(Technical Suppl)2017.
Artigo em Inglês | MEDLINE | ID: mdl-28736459

RESUMO

The comparison CCM.P-K15 is a key comparison in pressure involving six laboratories in three regional metrological organizations (RMO). The measurand of the comparison is the accommodation coefficient of two spinning rotating gauge characterized in nitrogen from 0.1 mPa up to 1.0 Pa. The two transfer standards were circulated from November 2009 until March 2011. The circulation consisted of three loops, one for each RMO, and a new calibration by the pilot between each loop. The stability of one of the transfer standards was poor and was worse than expected based on the previous history of the transfer standard while the other transfer standard demonstrated good stability while circulated in Europe and America and a fair stability while circulated in Asia. All the participants demonstrated equivalence to the definition of pressure in their respective primary standards.

18.
Molecules ; 22(3)2017 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-28245615

RESUMO

We previously reported that water-soluble cyclic selenides can mimic the antioxidative function of glutathione peroxidase (GPx) in water through a simple catalytic cycle, in which the selenide (>Se) is oxidized by H2O2 to the selenoxide (>Se=O) and the selenoxide is reduced by a thiol back to the selenide. In methanol, however, the GPx-like activity could not be explained by this simple scenario. To look into the reasons for the unusual behaviors in methanol, monoamino-substituted cyclic selenides with a variable ring size were synthesized, and the intermediates of the catalytic cycle were characterized by means of 77Se-NMR and LC-MS spectroscopies. In water, it was confirmed that the selenide and the selenoxide mainly contribute to the antioxidative function, though a slight contribution from the dihydroxy selenane (>Se(OH)2) was also suggested. In methanol, on the other hand, other active species, such as hydroxyselenonium (>Se⁺-OH) and hydroxy perhydroxy selenane (>Se(OH)(OOH)), could be generated to build another catalytic cycle. This over-oxidation would be more feasible for amino-substituted cyclic selenides, probably because the ammonium (NH3⁺) group would transfer a proton to the selenoxide moiety to produce a hydroxyselenonium species in the absence of an additional proton source. Thus, a shift of the major catalytic cycle in methanol would make the GPx-like antioxidative function of selenides perplexing.


Assuntos
Glutationa Peroxidase/metabolismo , Peróxido de Hidrogênio/química , Metanol/química , Compostos Organosselênicos/química , Catálise , Estrutura Molecular , Oxirredução
19.
Angew Chem Int Ed Engl ; 56(20): 5522-5526, 2017 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-28394477

RESUMO

Synthetic insulin analogues with a long lifetime are current drug targets for the therapy of diabetic patients. The replacement of the interchain disulfide with a diselenide bridge, which is more resistant to reduction and internal bond rotation, can enhance the lifetime of insulin in the presence of the insulin-degrading enzyme (IDE) without impairing the hormonal function. The [C7UA ,C7UB ] variant of bovine pancreatic insulin (BPIns) was successfully prepared by using two selenocysteine peptides (i.e., the C7U analogues of A- and B-chains, respectively). In a buffer solution at pH 10 they spontaneously assembled under thermodynamic control to the correct insulin fold. The selenoinsulin (Se-Ins) exhibited a bioactivity comparable to that of BPIns. Interestingly, degradation of Se-Ins with IDE was significantly decelerated (τ1/2 ≈8 h vs. ≈1 h for BPIns). The lifetime enhancement could be due to both the intrinsic stability of the diselenide bond and local conformational changes induced by the substitution.


Assuntos
Insulina/química , Insulina/síntese química , Sequência de Aminoácidos , Cristalografia por Raios X , Dissulfetos/química , Insulina/análogos & derivados , Modelos Moleculares
20.
Chembiochem ; 16(8): 1226-34, 2015 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-25881890

RESUMO

Fatty acid monoesters of the title compound (DHS(red) ), of variable carbon chain length (propionate, laurate, myristate, palmitate, and stearate), were synthesized, and their antioxidant capacities were evaluated by means of a lipid peroxidation assay with lecithin/cholesterol liposomes. The selenides with long alkyl chains exhibited significant antioxidant activity (IC50 =9-34 µM) against accumulation of lipid hydroperoxide. Incorporation of the myristate into the liposome was ≈50 % by EPMA analysis. Intermediacy of the selenoxide was examined by NMR. In addition, enhancement of interfacial redox catalytic activity was observed for the myristate, but not for PhSeSePh and edaravone, in a PhCl/H2 O biphasic peroxidation assay. These results suggested that a combination of a hydrophilic selenide moiety as a redox center with a long alkyl chain is an effective approach to selenium antioxidants with interfacial glutathione-peroxidase-like (GPx-like) activity. The activity can be controlled by the alkyl chain length.


Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Ácidos Graxos/química , Glicóis/química , Compostos Organosselênicos/química , Compostos Organosselênicos/farmacologia , Água/química , Colesterol/química , Colesterol/metabolismo , Radicais Livres/química , Interações Hidrofóbicas e Hidrofílicas , Lecitinas/química , Lecitinas/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipossomos/química , Lipossomos/metabolismo , Oxirredução , Solubilidade , Estereoisomerismo , Relação Estrutura-Atividade
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