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1.
Nat Commun ; 11(1): 4914, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-33004788

RESUMO

Oxepinamides are derivatives of anthranilyl-containing tripeptides and share an oxepin ring and a fused pyrimidinone moiety. To the best of our knowledge, no studies have been reported on the elucidation of an oxepinamide biosynthetic pathway and conversion of a quinazolinone to a pyrimidinone-fused 1H-oxepin framework by a cytochrome P450 enzyme in fungal natural product biosynthesis. Here we report the isolation of oxepinamide F from Aspergillus ustus and identification of its biosynthetic pathway by gene deletion, heterologous expression, feeding experiments, and enzyme assays. The nonribosomal peptide synthase (NRPS) OpaA assembles the quinazolinone core with D-Phe incorporation. The cytochrome P450 enzyme OpaB catalyzes alone the oxepin ring formation. The flavoenzyme OpaC installs subsequently one hydroxyl group at the oxepin ring, accompanied by double bond migration. The epimerase OpaE changes the D-Phe residue back to L-form, which is essential for the final methylation by OpaF.


Assuntos
Amidas/metabolismo , Aspergillus/enzimologia , Proteínas Fúngicas/metabolismo , Oxepinas/metabolismo , Amidas/química , Amidas/isolamento & purificação , Aspergillus/genética , Vias Biossintéticas , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Ensaios Enzimáticos , Proteínas Fúngicas/genética , Hidroxilação , Isomerismo , Metilação , Oxepinas/química , Oxepinas/isolamento & purificação , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Fenilalanina/química , Fenilalanina/metabolismo , Proteína O-Metiltransferase/genética , Proteína O-Metiltransferase/metabolismo , Quinazolinonas/metabolismo , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo
2.
J Environ Qual ; 49(5): 1435-1444, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33016451

RESUMO

Chlortetracycline (CTC), an antimicrobial administered as a feed additive to cattle, swine, and poultry, is present in the corresponding manure. Land application of raw or processed (composted or stockpiled) manure provides a mechanism by which CTC (and other antimicrobials) enters the environment and becomes available for transport to surface receiving waters via rainfall or snowmelt runoff. Chlortetracycline has been detected in Canadian surface waters, but little has been reported on its fate in aquatic ecosystems. To address this knowledge gap, the dissipation of CTC-enol was monitored in deionized water and water typical of wetlands within the prairie region of Canada. In deionized water, CTC-enol tautomerized to CTC-keto, and both tautomers epimerized to 4-epi-CTC-enol and 4-epi-CTC-keto, respectively. Irreversible isomerization to iso-CTC occurred, which then epimerized to 4-epi-iso-CTC. In wetland water, although tauterization of CTC-enol to CTC-keto occurred, there was no evidence of the formation of the 4-epimers of either CTC-enol or CTC-keto. The major product formed in the wetland water was iso-CTC, some of which epimerized to 4-epi-iso-CTC. Although CTC-enol was shown to tautomerize to CTC-keto, the concentration of CTC-keto remained low in both deionized and wetland water, suggesting that the isomerization of CTC-enol to iso-CTC most likely occurred via CTC-keto. The dissipation of CTC-enol in wetland water was described by pseudo first-order kinetics with a DT50 (time required for 50% dissipation) value of 4.8 h. The short DT50 value of CTC and reduced antimicrobial activity of iso-CTC and 4-epi-iso-CTC suggest a lower probability for selection for CTC-resistant bacteria in Canadian Prairie aquatic ecosystems.


Assuntos
Clortetraciclina , Animais , Canadá , Bovinos , Ecossistema , Pradaria , Isomerismo , Suínos , Áreas Alagadas
3.
Nat Commun ; 11(1): 4640, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934232

RESUMO

Small molecule inhibitors are prime reagents for studies in microtubule cytoskeleton research, being applicable across a range of biological models and not requiring genetic engineering. However, traditional chemical inhibitors cannot be experimentally applied with spatiotemporal precision suiting the length and time scales inherent to microtubule-dependent cellular processes. We have synthesised photoswitchable paclitaxel-based microtubule stabilisers, whose binding is induced by photoisomerisation to their metastable state. Photoisomerising these reagents in living cells allows optical control over microtubule network integrity and dynamics, cell division and survival, with biological response on the timescale of seconds and spatial precision to the level of individual cells within a population. In primary neurons, they enable regulation of microtubule dynamics resolved to subcellular regions within individual neurites. These azobenzene-based microtubule stabilisers thus enable non-invasive, spatiotemporally precise modulation of the microtubule cytoskeleton in living cells, and promise new possibilities for studying intracellular transport, cell motility, and neuronal physiology.


Assuntos
Microtúbulos/química , Paclitaxel/química , Linhagem Celular Tumoral , Citoesqueleto/química , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Humanos , Isomerismo , Microtúbulos/metabolismo , Neurônios/química , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Paclitaxel/farmacologia
4.
Proc Natl Acad Sci U S A ; 117(33): 19731-19736, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759207

RESUMO

Cyanobacteriochromes are photoreceptors in cyanobacteria that exhibit a wide spectral coverage and unique photophysical properties from the photoinduced isomerization of a linear tetrapyrrole chromophore. Here, we integrate femtosecond-resolved fluorescence and transient-absorption methods and unambiguously showed the significant solvation dynamics occurring at the active site from a few to hundreds of picoseconds. These motions of local water molecules and polar side chains are continuously convoluted with the isomerization reaction, leading to a nonequilibrium processes with continuous active-site motions. By mutations of critical residues at the active site, the modified local structures become looser, resulting in faster solvation relaxations and isomerization reaction. The observation of solvation dynamics is significant and critical to the correct interpretation of often-observed multiphasic dynamic behaviors, and thus the previously invoked ground-state heterogeneity may not be relevant to the excited-state isomerization reaction.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Fotorreceptores Microbianos/química , Proteínas de Bactérias/genética , Domínio Catalítico , Cianobactérias/química , Cianobactérias/genética , Isomerismo , Cinética , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo
5.
J Chromatogr A ; 1626: 461366, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32797845

RESUMO

An alternative method for efficient synthesis of urea-functionalized silanes was proposed on the basis of an N, N'-carbonyldiimidazole-mediated acyl-transfer reaction between various amino-containing building blocks. The employment of different parent aminosilanes and alkylamines afforded an array of urea-containing silanes, which were subsequently immobilized onto silica gel to form corresponding urea-embedded alkyl stationary phases for high-performance liquid chromatography. The different substituents on the silicon core of the derivatized silane were found to significantly influence the final chromatographic behaviors. The comparative chromatographic characterization of thus-prepared silica packings with conventional octadecyl (C18) stationary phases revealed that the urea group was beneficial to suppress silanol activity towards basic probes, as well as to increase the water-compatibility of the alkyl stationary phases. The combination of a polar urea moiety and a non-polar long alkyl chain was favorable for an enhanced steric selectivity towards shape-constrained isomers. The polarizability-sensitive feature of such stationary phases made them good candidates for efficient separation of nitro-containing polar substances.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Silanos/química , Ureia/química , Isomerismo , Silanos/síntese química , Sílica Gel/química , Dióxido de Silício/química
6.
J Oleo Sci ; 69(9): 1139-1143, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32788524

RESUMO

We compared the cytotoxic effects and tumor necrosis factor-α (TNF-α) production induced by 13 trans-octadecenoic acid positional isomers (trans-4-C18:1 to trans-16-C18:1) in RAW264.7 cells using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and enzyme-linked immunosorbent assay, respectively. No significant differences were observed in the cytotoxic effects among the 13 trans-C18:1 positional isomers and control on RAW264.7 cells. TNF-α production significantly decreased by treatment of trans-4-C18:1 as compared to control, but no significant differences in TNF-α production were observed among other trans-C18:1 positional isomers and control. These results suggest that the double bond position in trans-C18:1 may affect TNF-α production in cells.


Assuntos
Células RAW 264.7/metabolismo , Ácidos Esteáricos/toxicidade , Fator de Necrose Tumoral alfa/metabolismo , Animais , Isomerismo , Camundongos , Ácidos Esteáricos/química , Relação Estrutura-Atividade
7.
Anticancer Res ; 40(8): 4675-4680, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32727791

RESUMO

BACKGROUND: From the design and synthesis of enantiomers, we can expect to obtain two compounds with different pharmacokinetics and pharmacological activities at the same time, which is thought to lead to the development of efficient anticancer agents. Chiral-2-nitroimidazole TX-2036 derivatives exhibit stereo-configuration (R- and S-configuration)-dependent tyrosine kinase inhibitory activity, and the activity of the tyrosine kinase domain of EGF receptor (EGFR-tyk) is suppressed. In order to clarify the reason why the effects on EGFR-tyk activity differ depending on stereoisomers, we tried to analyze the interaction between each TX-2036 derivative and EGFR-tyk. MATERIALS AND METHODS: The 2-nitroimidazole-based radiosensitizer TX-2036 series were synthesized and their molecular features were examined using protein kinase inhibition assay and molecular structural analysis. RESULTS: R-configured TXs (TX-2043, -2030, and -2036) exhibited more potent protein kinase inhibitory activity than S-configured TXs (TX-2044, - 2031, and -2037), and the IC50 value of TX-2036 was 1.8 µM. CONCLUSION: R-configured TXs interacted with Lys721 and Thr766 of EGFR-tyk. The combinations of amino acid residues targeted by the S-configured TXs were different from each other (Ile765 and Thr766 (TX-2044), Ser696, Thr766, and Thr830 (TX-2031), Gly772, Cys773, and Thr830 (TX-2037)). Preparing a series of isomers with different target sites was considered beneficial when the target was mutated.


Assuntos
Domínios Proteicos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Isomerismo , Radiossensibilizantes/farmacologia , Estereoisomerismo
8.
J Chromatogr A ; 1625: 461332, 2020 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-32709358

RESUMO

Two structural isomeric pentacyclic triterpenes, oleanolic acid and ursolic acid, were considered as the models for the quality control of many traditional Chinese herbal medicines and they have been proved to own important pharmacological activities. In the present work, liquid chromatographic and liquid-liquid chromatographic separation with high peak resolution of structural isomeric oleanolic acid and ursolic acid using hydroxypropyl-ß-cyclodextrin as mobile phase additive was successfully achieved, respectively. A high peak resolution, RS=8.143, was achieved for the two structural isomeric compounds by conventional reverse phase high performance liquid chromatography, which was greatly improved compared with the published values. Meanwhile, a biphasic solvent system composed of n-hexane-ethyl acetate-0.1 mol/L hydroxypropyl-ß-cyclodextrin (9:1:10, v/v) was selected for liquid-liquid chromatography, which provided a high peak resolution, RS = 6.573, for analytical apparatus and Rs = 8.500 for semi-preparative apparatus after optimization by liquid-liquid extractions. Two elution modes including reverse phase mode and normal phase mode were investigated for preparative separation of two acids from crude exact of Eriobotrya japonica Thunb. Furthermore, the inclusion complex between each of the two structural isomers and hydroxypropyl-ß-cyclodextrin were also investigated for high performance liquid chromatography and liquid-liquid chromatography, respectively, in which formation constants were determined for oleanolic acid and ursolic acid.


Assuntos
2-Hidroxipropil-beta-Ciclodextrina/química , Cromatografia Líquida/métodos , Ácido Oleanólico/química , Ácido Oleanólico/isolamento & purificação , Triterpenos/química , Triterpenos/isolamento & purificação , Cromatografia de Fase Reversa , Eriobotrya/química , Isomerismo , Solventes , Temperatura , Termodinâmica , beta-Ciclodextrinas/química
9.
PLoS One ; 15(7): e0235863, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32706779

RESUMO

A comprehensive analysis of crystallographic data of 565 high-resolution protein homodimers comprised of over 250,000 residues suggests that amino acids form two groups that differ in their tendency to distort or symmetrize the structure of protein homodimers. Residues of the first group tend to distort the protein homodimer and generally have long or polar side chains. These include: Lys, Gln, Glu, Arg, Asn, Met, Ser, Thr and Asp. Residues of the second group contribute to protein symmetry and are generally characterized by short or aromatic side chains. These include: Ile, Pro, His, Val, Cys, Leu, Trp, Tyr, Phe, Ala and Gly. The distributions of the continuous symmetry measures of the proteins and the continuous chirality measures of their building blocks highlight the role of side chain geometry and the interplay between entropy and symmetry in dictating the conformational flexibility of proteins.


Assuntos
Aminoácidos/química , Conformação Proteica , Multimerização Proteica , Cristalografia por Raios X , Isomerismo
10.
Chemosphere ; 260: 127607, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32698116

RESUMO

Diet is considered a major route of human exposure to hexabromocyclododecane, a chiral environmental contaminant. A previous study reported on the occurrence of hexabromocyclododecane diastereoisomers in food items of animal origin collected in Belgium. The present study reports further results on corresponding enantiomeric fractions of the same samples. None of the samples could be considered as racemic for the α-isomer suggesting that foodstuff contamination occurred prior to death of the corresponding producing animal and was not the result of the food item being in contact with technical HBCDD. Non-racemic chiral signatures were also observed for ß- and γ-isomers. We conclude that, depending on their dietary habits, different individuals might be overall exposed to non-racemic profiles. Considering that toxicological effects are enantiomer-dependent, this could modulate potential adverse effects.


Assuntos
Poluentes Ambientais , Contaminação de Alimentos , Alimentos , Hidrocarbonetos Bromados/análise , Animais , Bélgica , Humanos , Isomerismo , Estereoisomerismo
11.
Food Chem ; 333: 127499, 2020 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-32673957

RESUMO

This study was to examine the formation of advanced glycation end-products (AGEs) in black tea during drying process at 90, 120, and 150 °C for 1 h. Nine AGEs including Nɛ-(carboxyethyl)-l-lysine (CEL), Nɛ-(carboxymethyl)-l-lysine (CML), three isomers of methylglyoxal-hydroimidazolones (MG-Hs), three isomers of glyoxal-hydroimidazolones (GO-Hs), and argpyrimidine were quantified by using HPLC-MS/MS with isotope-labelled internal standard. Results showed that each AGE during the drying process of 150 °C was significantly higher (p < 0.05) than at 90 and 120 °C, and argpyrimidine was only found in the treatment of 150 °C. MG-H1/3 was first quantified as the major AGE during drying at 120-150 °C, the content respectively reached to (39.66 ± 2.61) µg/g and (58.88 ± 1.76) µg/g after 1 h drying, where CML content only had (19.86 ± 1.02) µg/g and (23.71 ± 1.40) µg/g. This study indicated that arginine derived-AGEs are the key components of black tea AGEs.


Assuntos
Camellia sinensis/química , Produtos Finais de Glicação Avançada/análise , Imidazóis/química , Lisina/análogos & derivados , Extratos Vegetais/química , Aldeído Pirúvico/química , Cromatografia Líquida de Alta Pressão , Dessecação , Manipulação de Alimentos , Humanos , Isomerismo , Lisina/química , Folhas de Planta/química , Espectrometria de Massas em Tandem , Chá
12.
Nature ; 583(7815): 314-318, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32499654

RESUMO

Light-driven sodium pumps actively transport small cations across cellular membranes1. These pumps are used by microorganisms to convert light into membrane potential and have become useful optogenetic tools with applications in neuroscience. Although the resting state structures of the prototypical sodium pump Krokinobacter eikastus rhodopsin 2 (KR2) have been solved2,3, it is unclear how structural alterations over time allow sodium to be translocated against a concentration gradient. Here, using the Swiss X-ray Free Electron Laser4, we have collected serial crystallographic data at ten pump-probe delays from femtoseconds to milliseconds. High-resolution structural snapshots throughout the KR2 photocycle show how retinal isomerization is completed on the femtosecond timescale and changes the local structure of the binding pocket in the early nanoseconds. Subsequent rearrangements and deprotonation of the retinal Schiff base open an electrostatic gate in microseconds. Structural and spectroscopic data, in combination with quantum chemical calculations, indicate that a sodium ion binds transiently close to the retinal within one millisecond. In the last structural intermediate, at 20 milliseconds after activation, we identified a potential second sodium-binding site close to the extracellular exit. These results provide direct molecular insight into the dynamics of active cation transport across biological membranes.


Assuntos
Flavobacteriaceae/química , Rodopsinas Microbianas/química , Rodopsinas Microbianas/efeitos da radiação , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/efeitos da radiação , Sítios de Ligação , Cristalografia , Elétrons , Transporte de Íons , Isomerismo , Lasers , Prótons , Teoria Quântica , Retinaldeído/química , Retinaldeído/metabolismo , Bases de Schiff/química , Sódio/metabolismo , Análise Espectral , Eletricidade Estática , Fatores de Tempo
13.
Environ Pollut ; 264: 114747, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32559878

RESUMO

Tricresyl phosphates (TCPs), as representative aromatic organophosphate flame retardants (OPFRs), have received much attention due to their potential neurotoxicity and endocrine-disrupting effects. However, the role of estrogen receptor α (ERα) and G protein-coupled estrogen receptor (GPER) in their estrogen disrupting effects remains poorly understood. Therefore, in this study, three TCP isomers, tri-o-cresyl phosphate (ToCP), tri-m-cresyl phosphate (TmCP) and tri-p-cresyl phosphate (TpCP), were examined for their activities on ERα by using two-hybrid yeast assay, and action on GPER by using Boyden chamber assay, cAMP production assay, calcium mobilization assay and molecular docking analysis. The results showed that three TCP isomers were found to act as ERα antagonists. Conversely, they had agonistic activity on GPER to promote GPER-mediated cell migration of MCF7 cells and SKBR3 cells. Both ToCP and TpCP activated GPER-mediated cAMP production and calcium mobilization, whereas TmCP had different mode of action, it only triggered GPER-mediated calcium mobilization, as evidenced by using the specific GPER inhibitor (G15) and GPER overexpressing experiments. Molecular docking further revealed that the way of interaction of TmCP and TpCP with GPER was different from that of ToCP with GPER, and higher activity of ToCP in activating GPER-mediated pathways might be associated with the alkyl substitution at the ortho position of the aromatic ring. Our results, for the first time, found a new target, GPER, for TCPs exerting their estrogen-disrupting effects, and demonstrated complex estrogen-disrupting effects of three TCP isomers involved their opposite activities toward ERα and GPER.


Assuntos
Tritolil Fosfatos , Estrogênios , Humanos , Isomerismo , Simulação de Acoplamento Molecular , Transdução de Sinais
14.
Food Chem ; 330: 127209, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32535314

RESUMO

Bovine ß-casein is an amphiphilic protein that exists as a monomer and self-organizes into micelles in aqueous solution. The protein has been used as natural vehicles for bioactives. Trans-resveratrol has received significant attention due to its vast health benefits and conversion to cis-isomer during processing and storage. However, cis-isomer has not yet gained as much attention as that of trans-isomer. In this study, the interaction of ß-casein with trans- and cis-resveratrol was characterized. Trans-resveratrol exhibited a higher affinity for ß-casein than cis-isomer, and ß-casein could bind two isomers simultaneously to form protein-diligand complexes. Both trans- and cis-isomers could be encapsulated into ß-casein micelles with encapsulation efficiencies of ~69% and ~57%, respectively. The ß-casein micelles could delay photo-isomerization of trans-isomer to cis-isomer, while ß-casein-ligand complex showed a better protective effect for both isomers during storage than ß-casein micelles. These results might be useful for the development of protein-based carriers for the polyphenols.


Assuntos
Caseínas/química , Excipientes/química , Resveratrol/química , Animais , Bovinos , Isomerismo , Ligantes , Micelas
15.
Proc Natl Acad Sci U S A ; 117(28): 16356-16362, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32591422

RESUMO

Phytochromes are a diverse family of bilin-binding photoreceptors that regulate a wide range of physiological processes. Their photochemical properties make them attractive for applications in optogenetics and superresolution microscopy. Phytochromes undergo reversible photoconversion triggered by the Z ⇄ E photoisomerization about the double bond in the bilin chromophore. However, it is not fully understood at the molecular level how the protein framework facilitates the complex photoisomerization dynamics. We have studied a single-domain bilin-binding photoreceptor All2699g1 (Nostoc sp. PCC 7120) that exhibits photoconversion between the red light-absorbing (Pr) and far red-absorbing (Pfr) states just like canonical phytochromes. We present the crystal structure and examine the photoisomerization mechanism of the Pr form as well as the formation of the primary photoproduct Lumi-R using time-resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations. We show that the unusually long excited state lifetime (broad lifetime distribution centered at ∼300 picoseconds) is due to the interactions between the isomerizing pyrrole ring D and an adjacent conserved Tyr142. The decay kinetics shows a strongly distributed character which is imposed by the nonexponential protein dynamics. Our findings offer a mechanistic insight into how the quantum efficiency of the bilin photoisomerization is tuned by the protein environment, thereby providing a structural framework for engineering bilin-based optical agents for imaging and optogenetics applications.


Assuntos
Fitocromo/química , Fitocromo/metabolismo , Pigmentos Biliares/química , Pigmentos Biliares/metabolismo , Cristalografia por Raios X , Isomerismo , Cinética , Modelos Moleculares , Nostoc/metabolismo , Processos Fotoquímicos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Conformação Proteica , Análise Espectral , Relação Estrutura-Atividade
16.
Toxicon ; 184: 175-179, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32585217

RESUMO

There is evidence that the environmental toxin ß-N-methylamino-L-alanine (L-BMAA) may be involved in neurodegenerative diseases. However, a number of controversies exist regarding L-BMAA, one of which is the possibility that when assaying for L-BMAA, its isomers are being detected instead. There are at least four isomers of BMAA that are known to occur: L-BMAA, ß-N-methylamino-D-alanine (D-BMAA), 2,4-diaminobutyric acid (DAB), and N-(2-aminoethyl)glycine (AEG). The fact that isomers of BMAA exist in nature also leads to the possibility that they are involved in toxicity. We set out to determine both the potency and the mechanism of toxicity of L-BMAA, D-BMAA, DAB, asnd AEG using primary cortical cultures. The results were surprising with the following order of potency of toxicity: AEG > DAB > D-BMAA > L-BMAA. These results suggest that AEG may be an overlooked neurotoxin. We found that AEG induced toxicity through mGluR5 receptors and induction of oxidative stress. While the potential role of L-BMAA in neurodegenerative diseases has been emphasized, other isomers of L-BMAA, particularly AEG, are actually more potent toxins, and could therefore potentially contribute to neurodegenerative diseases.


Assuntos
Diamino Aminoácidos/toxicidade , Agonistas de Aminoácidos Excitatórios/toxicidade , Animais , Monitoramento Ambiental , Glicina , Isomerismo , Síndromes Neurotóxicas , Neurotoxinas , Espectrometria de Massas em Tandem
17.
Biochim Biophys Acta Proteins Proteom ; 1868(9): 140459, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32474105

RESUMO

In the biological proteins, aspartic acid (Asp) residues are prone to nonenzymatic isomerization via a succinimide (Suc) intermediate. Asp-residue isomerization causes the aggregation and the insolubilization of proteins, and is considered to be involved in various age-related diseases. Although Suc intermediate was considered to be formed by nucleophilic attack of the main-chain amide nitrogen of N-terminal side adjacent residue to the side-chain carboxyl carbon of Asp residue, previous studies have shown that the nucleophilic attack is more likely to proceed via iminol tautomer when the water molecules act as catalysts. However, the full pathway to Suc-intermediate formation has not been investigated, and the experimental analyses for the Asp-residue isomerization mechanism at atomic and molecular levels, such as the analysis of the transition state geometry, are difficult. In the present study, we computationally explored the full pathways for Suc-intermediate formation from Asp residues. The calculations were performed two types of reactant complexes, and all energy minima and TS geometries were optimized using B3LYP density functional methods. As a result, the SI-intermediate formation was divided into three processes, i.e., iminolization, cyclization, and dehydration processes, and the activation energies were calculated to be 26.1 or 28.4 kcal mol-1. These values reproduce the experimental data. The computational results show that abundant water molecules in living organisms are effective catalysts for the Asp-residue isomerization.


Assuntos
Ácido Aspártico/química , Modelos Químicos , Succinimidas/síntese química , Água/química , Amidas , Catálise , Ciclização , Isomerismo , Modelos Moleculares , Nitrogênio , Proteínas/química
18.
Nat Commun ; 11(1): 2827, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499507

RESUMO

Understanding the connection between the motion of the nuclei in a molecule and the rearrangement of its electrons lies at the heart of chemistry. While many experimental methods have been developed to probe either the electronic or the nuclear structure on the timescale of atomic motion, very few have been able to capture both these changes in concert. Here, we use time-resolved photoelectron imaging to probe the isomerisation coordinate on the excited state of an isolated model chromophore anion of the photoactive yellow protein. By probing both the electronic structure changes as well as nuclear dynamics, we are able to uniquely measure isomerisation about a specific bond. Our results demonstrate that the photoelectron signal dispersed in time, energy and angle combined with calculations can track the evolution of both electronic and geometric structure along the adiabatic state, which in turn defines that chemical transformation.


Assuntos
Proteínas de Bactérias/química , Elétrons , Fotorreceptores Microbianos/química , Anisotropia , Isomerismo , Fatores de Tempo
19.
Nat Commun ; 11(1): 2460, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424138

RESUMO

Fluorescent barcoding is a pivotal technique for the investigation of the microscale world, from information storage to the monitoring of dynamic biochemical processes. Using fluorescence lifetime as the readout modality offers more reproducible and quantitative outputs compared to conventional fluorescent barcoding, being independent of sample concentration and measurement methods. However, the use of fluorescence lifetime in this area has been limited by the lack of strategies that provide spatiotemporal manipulation of the coding process. In this study, we design a two-component photo-switchable nanogel that exhibits variable fluorescence lifetime upon photoisomerization-induced energy transfer processes through light irradiation. This remotely manipulated fluorescence lifetime property could be visually mapped using fluorescence lifetime imaging microscopy (FLIM), allowing selective storage and display of information at the microscale. Most importantly, the reversibility of this system further provides a strategy for minimizing the background influence in fluorescence lifetime imaging of live cells and sub-cellular organelles.


Assuntos
Luz , Microscopia de Fluorescência/métodos , Células A549 , Sobrevivência Celular , Transferência de Energia , Fluorescência , Humanos , Isomerismo , Mitocôndrias/metabolismo , Nanogéis/química , Polietilenoglicóis/química , Polietilenoimina/química , Polímeros/química , Frações Subcelulares
20.
Nat Biomed Eng ; 4(5): 560-571, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32393891

RESUMO

The oral administration of peptide drugs is hampered by their metabolic instability and limited intestinal uptake. Here, we describe a method for the generation of small target-specific peptides (less than 1,600 Da in size) that resist gastrointestinal proteases. By using phage display to screen large libraries of genetically encoded double-bridged peptides on protease-resistant fd bacteriophages, we generated a peptide inhibitor of the coagulation Factor XIa with nanomolar affinity that resisted gastrointestinal proteases in all regions of the gastrointestinal tract of mice after oral administration, enabling more than 30% of the peptide to remain intact, and small quantities of it to reach the blood circulation. We also developed a gastrointestinal-protease-resistant peptide antagonist for the interleukin-23 receptor, which has a role in the pathogenesis of Crohn's disease and ulcerative colitis. The de novo generation of targeted peptides that resist proteolytic degradation in the gastrointestinal tract should help the development of effective peptides for oral delivery.


Assuntos
Peptídeos/administração & dosagem , Peptídeos/uso terapêutico , Proteólise , Administração Oral , Sequência de Aminoácidos , Animais , Técnicas de Visualização da Superfície Celular , Cristalografia por Raios X , Feminino , Trato Gastrointestinal/metabolismo , Humanos , Isomerismo , Camundongos Endogâmicos BALB C , Modelos Moleculares , Peptídeo Hidrolases/metabolismo , Biblioteca de Peptídeos , Peptídeos/química , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptores de Interleucina/antagonistas & inibidores , Receptores de Interleucina/metabolismo
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