RESUMO
Human relaxin-2 (H2 relaxin) is a peptide hormone of about 6 kDa, first identified as a reproductive hormone involved in vasoregulation during pregnancy. It has recently attracted strong interest because of its diverse functions, including anti-inflammatory, anti-fibrotic, and vasodilatory, and has been suggested as a potential peptide-based drug candidate for a variety of diseases. Mature H2 relaxin is constituted by the A- and B-chains stabilized by two interchain disulfide (SS) bridges and one intrachain SS linkage. In this study, seleno-relaxins, SeRlx-α and SeRlx-ß, which are [C11UA,C11UB] and [C10UA,C15UA] variants of H2 relaxin, respectively, were synthesized via a one-pot oxidative chain assembly (folding) from the component A- and B-chains. The substitution of SS bonds in a protein with their analogue, diselenide (SeSe) bonds, has been shown to alter the physical, chemical, and physiological properties of the protein. The surface SeSe bond (U11A-U11B) enhanced the yield of chain assembly while the internal SeSe bond (U10A-U15A) improved the reaction rate of the folding, indicating that these bridges play a major role in controlling the thermodynamics and kinetics, respectively, of the folding mechanism. Furthermore, SeRlx-α and SeRlx-ß effectively reduced the expression of a tissue fibrosis-related factor in human endometriotic stromal cells. Thus, the findings of this study indicate that the S-to-Se substitution strategy not only enhances the foldability of relaxin, but also provides new guidance for the development of novel relaxin formulations for endometriosis treatment.
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/químicaRESUMO
Compounds harboring high acidity and oxidizability of thiol groups permit tuning the redox equilibrium constants of CxxC sites of members of the protein disulphide isomerase (PDI) family and thus can be used to accelerate folding processes and increase the production of native proteins by minimal loading in comparison to glutathione.
Assuntos
Isomerases de Dissulfetos de Proteínas , Isomerases de Dissulfetos de Proteínas/metabolismo , Isomerases de Dissulfetos de Proteínas/química , Oxirredução , Dobramento de Proteína , Motivos de Aminoácidos , Humanos , Glutationa/metabolismo , Glutationa/químicaRESUMO
Seleno-insulin, a class of artificial insulin analogs, in which one of the three disulfide-bonds (S-S's) of wild-type insulin (Ins) is replaced by a diselenide-bond (Se-Se), is attracting attention for its unique chemical and physiological properties that differ from those of Ins. Previously, we pioneered the development of a [C7UA,C7UB] analog of bovine pancreatic insulin (SeIns) as the first example, and demonstrated its high resistance against insulin-degrading enzyme (IDE). In this study, the conditions for the synthesis of SeIns via native chain assembly (NCA) were optimized to attain a maximum yield of 72%, which is comparable to the in vitro folding efficiency for single-chain proinsulin. When the resistance of BPIns to IDE was evaluated in the presence of SeIns, the degradation rate of BPIns became significantly slower than that of BPIns alone. Furthermore, the investigation on the intermolecular association properties of SeIns and BPIns using analytical ultracentrifugation suggested that SeIns readily forms oligomers not only with its own but also with BPIns. The hypoglycemic effect of SeIns on diabetic rats was observed at a dose of 150 µg/300 g rat. The strategy of replacing the solvent-exposed S-S with Se-Se provides new guidance for the design of long-acting insulin formulations.
RESUMO
We report the first example of a synthetic thiol-based compound that promotes oxidative protein folding upon 1-equivalent loading to the disulfide bonds in the client protein to afford the native form in over 70% yield. N-Methylation is a central post-translational processing of proteins in vivo for regulating functions including chaperone activities. Despite the universally observed biochemical reactions in nature, N-methylation has hardly been utilized in the design, functionalization, and switching of synthetic bioregulatory agents, particularly folding promotors. As a biomimetic approach, we developed pyridinylmethanethiols to investigate the effects of N-methylation on the promotion of oxidative protein folding. For a comprehensive study on the geometrical effects, constitutional isomers of pyridinylmethanethiols with ortho-, meta-, and para-substitutions have been synthesized. Among the constitutional isomers, para-substituted pyridinylmethanethiol showed the fastest disulfide-bond formation of the client proteins to afford the native forms most efficiently. N-Methylation drastically increased the acidity and enhanced the oxidizability of the thiol groups in the pyridinylmethanethiols to enhance the folding promotion efficiencies. Among the isomers, para-substituted N-methylated pyridinylmethanethiol accelerated the oxidative protein folding reactions with the highest efficiency, allowing for protein folding promotion by 1-equivalent loading as a semi-enzymatic activity. This study will offer a novel bioinspired molecular design of synthetic biofunctional agents that are semi-enzymatically effective for the promotion of oxidative protein folding including biopharmaceuticals such as insulin in vitro by minimum loading.
RESUMO
Systemically administered lipid nanoparticles (LNPs) are complexed with Apolipoprotein E (ApoE) in the bloodstream, and the complex is subsequently largely taken up by hepatocytes. Based on a previous report showing that, like blood, lymph fluid also contains ApoE, and that LECs, in turn, expresses a low density-lipoprotein receptor (LDLR), which is the receptor responsible for the ApoE-bound LNP, we hypothesized that subcutaneously administered LNPs would be taken up by LECs via an ApoE-LDLR pathway. Our in vitro studies using immortal LECs that we established in a previous study showed that LEC indeed took up LNPs in an ApoE-dependent manner. We then reported on the development of LNPs that target the lymphatic endothelium for in vivo siRNA delivery after subcutaneous administration. The key to success for in vivo LEC targeting is that the surface needs to be modified with a high density of polyethylene glycol (PEG)-conjugated lipids with short acyl chains (C14). The LNPs were drained into the lymphatic system, and then accumulated in lymphatic endothelial cells in an ApoE-dependent manner, most likely after the release of the PEG-lipid. Subcutaneous administration of optimized LNPs containing encapsulated siRNA against VEGFR3, a marker of LECs, significantly inhibited the expression of VEGFR3. These findings are the first report of a simple straightforward strategy for targeting lymphatic endothelial cells by using ionizable lipid-formulated LNPs.
Assuntos
Células Endoteliais , Nanopartículas , RNA Interferente Pequeno/metabolismo , Células Endoteliais/metabolismo , Apolipoproteínas E/metabolismo , Lipídeos , Polietilenoglicóis/metabolismoRESUMO
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 Seâ â â 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 Seâ â â 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/químicaRESUMO
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 NHâ â â 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 SulfidrilaRESUMO
In a previous study, we reported that (S)-1,2-diselenane-4-amine (1) catalyzes oxidative protein folding through protein disulfide isomerase (PDI)-like catalytic mechanisms and that the direct conjugation of a basic amino acid (Xaa: His, Lys, or Arg) via an amide bond improves the catalytic activity of 1 by increasing its diselenide (Se-Se) reduction potential (E'°). In this study, to modulate the Se-Se redox properties and the association of the compounds with a protein substrate, new catalysts, in which a Gly spacer was inserted between 1 and Xaa, were synthesized. Exhaustive comparison of the PDI-like catalytic activities and E'° values among 1, 1-Xaa, and 1-Gly-Xaa showed that the insertion of a Gly spacer into 1-Xaa either did not change or slightly reduced the PDI-like activity and the E'° values. Importantly, however, only 1-Gly-Arg deviated from this generality and showed obviously increased E°' value and PDI-like activity compared to the corresponding compound with no Gly spacer (1-Arg); on the contrary, its catalytic activity was the highest among the diselenide compounds employed in this study, while this abnormal enhancement of the catalytic activity of 1-Gly-Arg could not be fully explained by the thermodynamics of the Se-Se bond and its association ability with protein substrates.
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)/metabolismoRESUMO
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 ProteínaRESUMO
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 CultivadasRESUMO
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/farmacologiaRESUMO
BACKGROUND: Relaxin (RLX)-2, produced by the corpus luteum and placenta, is known to be potentially effective in fibrotic diseases of the heart, lungs, kidneys, and bladder; however, its effectiveness in endometriosis has not yet been investigated. In the present study, we conducted a comprehensive study on the effect of RLX-2 on endometriosis. We checked the expressions of LGR-7, a primary receptor of RLX-2, in endometriomas using immunohistochemistry. Endometriotic stromal cells (ESCs) purified from surgical specimens were used in in vitro experiments. The effects of RLX-2 on ESCs were evaluated by quantitative-PCR, ELISA, and Western blotting. Gel contraction assay was used to assess the contraction suppressive effect of RLX-2. The effect of RLX-2 was also examined in the endometriosis mouse model. LGR-7 was expressed in endometriotic lesions. In ESCs, RLX-2 increased the production of cAMP and suppressed the secretion of interleukin-8, an inflammatory cytokine, by 15% and mRNA expression of fibrosis-related molecules, plasminogen activator inhibitor-1 (PAI-1), and collagen-I by approximately 50% (p < 0.05). In the gel contraction assay, RLX-2 significantly suppressed the contraction of ESCs, which was cancelled by removing RLX-2 from the medium or by adding H89, a Protein Kinase A (PKA) inhibitor. In ESCs stimulated with RLX-2, p38 MAPK phosphorylation was significantly suppressed. In the endometriosis mouse model, administration of RLX-2 significantly decreased the area of the endometriotic-like lesion with decreasing fibrotic component compared to non-treated control (p = 0.01). RLX-2 may contribute to the control of endometriotic lesion by suppressing fibrosis, scar formation, and inflammation.
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 , TranscriptomaRESUMO
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/metabolismoRESUMO
Protein disulfide isomerase (PDI) can assist immature proteins to correctly fold by controlling cysteinyl disulfide (SS)-relating reactions (i. e., SS-formation, SS-cleavage, and SS-isomerization). PDI controls protein quality by suppressing protein aggregation, as well as functions as an oxidative folding catalyst. Following the amino acid sequence of the active center in PDI, basic amino acid conjugates of 1,2-diselenan-4-amine (1), which show oxidoreductase- and isomerase-like activities for SS-relating reactions, were designed as a novel PDI model compound. By conjugating the amino acids, the diselenide reduction potential of compound 1 was significantly increased, causing improvement of the catalytic activities for all SS-relating reactions. Furthermore, these compounds, especially histidine-conjugated one, remarkably suppressed protein aggregation even at low concertation (0.3â mMâ¼). Thus, it was demonstrated that the conjugation of basic amino acids into 1 simultaneously achieves the enhancement of the redox reactivity and the capability to suppress protein aggregation.
Assuntos
Aminoácidos Básicos/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Aminoácidos Básicos/química , Humanos , Conformação Molecular , Agregados Proteicos , Isomerases de Dissulfetos de Proteínas/química , Controle de QualidadeRESUMO
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 , RatosRESUMO
A dirhodium-catalyzed, ß-selective C-H amination of organosilicon compounds has been developed. Primary C(sp3)-H bonds of silylethyl groups and secondary C(sp3)-H bonds of silacycloalkanes can be selectively converted to C-N bonds at the ß-position of the silicon atoms. The experimental data and theoretical calculations indicate that the strong σ-donor ability of the carbon-silicon bonds is responsible for the ß-selectivity. Kinetic isotope effects clearly demonstrate that the C-H bond cleavage step is not turnover-limiting, but selectivity-determining.
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.