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1.
Int J Nanomedicine ; 14: 6339-6356, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496690

RESUMO

Objective: The rational combination of immunotherapy with standard chemotherapy shows synergistic clinical activities in cancer treatment. In the present study, an oral powder formulation of pemetrexed (PMX) was developed to enhance intestinal membrane permeability and investigate its application in metronomic chemotherapy in combination with immunotherapy. Methods: PMX was ionically complexed with a bile acid derivative (Nα-deoxycholyl-l-lysyl-methylester; DCK) as a permeation enhancer and mixed with dispersing agents, such as poloxamer 188 (P188) and Labrasol, to form an amorphous oral powder formulation of PMX/DCK (PMX/DCK-OP). Results: The apparent permeability (Papp) of PMX/DCK-OP across a Caco-2 cell monolayer was 2.46- and 8.26-fold greater than that of PMX/DCK and free PMX, respectively, which may have been due to the specific interaction of DCK with bile acid transporters, as well as the alteration of membrane fluidity due to Labrasol and P188. Furthermore, inhibition of bile acid transporters by actinomycin D in Caco-2 cell monolayers decreased the Papp of PMX/DCK-OP by 75.4%, suggesting a predominant role of bile acid transporters in the intestinal absorption of PMX/DCK-OP. In addition, caveola/lipid raft-dependent endocytosis, macropinocytosis, passive diffusion, and paracellular transport mechanisms significantly influenced the permeation of PMX/DCK-OP through the intestinal membrane. Therefore, the oral bioavailability of PMX/DCK-OP in rats was 19.8%±6.93%, which was 294% higher than that of oral PMX. Moreover, an in vivo anticancer efficacy study in B16F10 cell-bearing mice treated with a combination of oral PMX/DCK-OP and intraperitoneal anti-PD1 exhibited significant suppression of tumor growth, and the tumor volume was maximally inhibited by 2.03- and 3.16-fold compared to the oral PMX/DCK-OP and control groups, respectively. Conclusion: These findings indicated the therapeutic potential of a combination of low-dose oral chemotherapy and immunotherapy for synergistic anticancer efficacy.


Assuntos
Ácido Desoxicólico/química , Composição de Medicamentos , Intestinos/efeitos dos fármacos , Pemetrexede/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/sangue , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Disponibilidade Biológica , Transporte Biológico/efeitos dos fármacos , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/química , Humanos , Íons , Lisina/análogos & derivados , Lisina/química , Camundongos Endogâmicos BALB C , Pemetrexede/administração & dosagem , Pemetrexede/sangue , Pemetrexede/farmacocinética , Permeabilidade , Ratos Sprague-Dawley
2.
J Agric Food Chem ; 67(36): 10214-10221, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31430143

RESUMO

5-Hydroxymethylfurfural (HMF) can undergo the Maillard reaction with amino acids. However, the safety of the products remains unknown. In this study, a HMF-lysine Schiff base named (E)-N6-((5'-(hydroxymethyl)furan-2'-yl)methylene)lysine (HML) was identified and detected for the first time in baked foods. HML formation significantly decreased the cytotoxicity (IC50) of HMF against GES-1 cells (81.81 versus 5.02 mM and 73.76 versus 2.94 mM for HML versus HMF at 24 and 48 h, respectively), EA.hy926 cells (86.05 versus 4.85 mM and 77.22 versus 0.71 mM, respectively), and Caco-2 cells (155.77 versus 36.84 mM and 112.70 versus 18.51 mM, respectively). Exposure of Caco-2 cells to HMF at 10.0 mM triggered cell apoptosis of 14.02% (versus 8.54% in the control), whereas exposure to HML at 10-15 mM hardly increased cell apoptosis. Moreover, the absorption capacities of HMF and HML by Caco-2 cells were equivalent (p > 0.05) at 7.23-12.57% after incubation at 2 mM for 30-150 min.


Assuntos
Furaldeído/análogos & derivados , Lisina/química , Apoptose/efeitos dos fármacos , Linhagem Celular , Furaldeído/química , Furaldeído/toxicidade , Humanos , Lisina/toxicidade , Reação de Maillard
3.
Chem Commun (Camb) ; 55(67): 9979-9982, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31367719

RESUMO

A chemical tag enhances peptide detection by multiple orders in mass spectrometry. The substantial improvement in the peptide mapping along with simplified and enhanced fragmentation pattern enables the unambiguous sequencing of a protein and antibody. The chemoselective sensitivity booster provides a tool for remarkably improved analysis of protein bioconjugates.


Assuntos
Peptídeos/análise , Proteínas/análise , Citocromos c/análise , Lisina/química , Mapeamento de Peptídeos , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem
4.
Nat Commun ; 10(1): 2909, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266957

RESUMO

Cells form and use biomolecular condensates to execute biochemical reactions. The molecular properties of non-membrane-bound condensates are directly connected to the amino acid content of disordered protein regions. Lysine plays an important role in cellular function, but little is known about its role in biomolecular condensation. Here we show that protein disorder is abundant in protein/RNA granules and lysine is enriched in disordered regions of proteins in P-bodies compared to the entire human disordered proteome. Lysine-rich polypeptides phase separate into lysine/RNA-coacervates that are more dynamic and differ at the molecular level from arginine/RNA-coacervates. Consistent with the ability of lysine to drive phase separation, lysine-rich variants of the Alzheimer's disease-linked protein tau undergo coacervation with RNA in vitro and bind to stress granules in cells. Acetylation of lysine reverses liquid-liquid phase separation and reduces colocalization of tau with stress granules. Our study establishes lysine as an important regulator of cellular condensation.


Assuntos
Lisina/metabolismo , RNA/química , RNA/metabolismo , Proteínas tau/química , Proteínas tau/metabolismo , Acetilação , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Linhagem Celular , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Humanos , Lisina/química , Lisina/genética , RNA/genética , Proteínas tau/genética
5.
J Agric Food Chem ; 67(28): 7821-7831, 2019 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-31260293

RESUMO

The mechanism of inhibition of advanced glycation end product (AGE) formation by protocatechuic acid and 3,4-dihydroxyphenylacetic acid (DHPA) has been studied using a widespread applied in vitro model system composed of bovine serum albumin (BSA) and supraphysiological glucose concentrations. Protocatechuic acid and DHPA inhibited the formation of Amadori compounds, fluorescent AGEs (IC50 = 62.1 ± 1.4 and 155.4 ± 1.1 µmol/L, respectively), and Nε-(carboxymethyl)lysine (IC50 = 535.3 ± 1.1 and 751.2 ± 1.0 µmol/L, respectively). BSA was pretreated with the two phenolic acids, and the formation of BSA-phenolic acid adducts was estimated by nanoflow liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry. Results showed that the tested phenolic acids bound key sites of glycation in BSA through a metal-catalyzed oxidative mechanism. The antiglycative activity mechanism involved the formation of BSA-phenolic acid adducts, and it is unlikely that this occurs in vivo. These results raise the problem to design in vitro models closer to physiological conditions to reach biologically sound conclusions.


Assuntos
Ácido 3,4-Di-Hidroxifenilacético/química , Hidroxibenzoatos/química , Lisina/química , Metais/química , Soroalbumina Bovina/química , Animais , Catálise , Bovinos , Cromatografia Líquida , Glicosilação , Oxirredução , Espectrometria de Massas por Ionização por Electrospray
6.
PLoS Genet ; 15(6): e1008196, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31173582

RESUMO

Covalent intermolecular cross-linking of collagen is essential for tissue stability. Recent studies have demonstrated that cyclophilin B (CypB), an endoplasmic reticulum (ER)-resident peptidyl-prolyl cis-trans isomerase, modulates lysine (Lys) hydroxylation of type I collagen impacting cross-linking chemistry. However, the extent of modulation, the molecular mechanism and the functional outcome in tissues are not well understood. Here, we report that, in CypB null (KO) mouse skin, two unusual collagen cross-links lacking Lys hydroxylation are formed while neither was detected in wild type (WT) or heterozygous (Het) mice. Mass spectrometric analysis of type I collagen showed that none of the telopeptidyl Lys was hydroxylated in KO or WT/Het mice. Hydroxylation of the helical cross-linking Lys residues was almost complete in WT/Het but was markedly diminished in KO. Lys hydroxylation at other sites was also lower in KO but to a lesser extent. A key glycosylation site, α1(I) Lys-87, was underglycosylated while other sites were mostly overglycosylated in KO. Despite these findings, lysyl hydroxylases and glycosyltransferase 25 domain 1 levels were significantly higher in KO than WT/Het. However, the components of ER chaperone complex that positively or negatively regulates lysyl hydroxylase activities were severely reduced or slightly increased, respectively, in KO. The atomic force microscopy-based nanoindentation modulus were significantly lower in KO skin than WT. These data demonstrate that CypB deficiency profoundly affects Lys post-translational modifications of collagen likely by modulating LH chaperone complexes. Together, our study underscores the critical role of CypB in Lys modifications of collagen, cross-linking and mechanical properties of skin.


Assuntos
Ciclofilinas/química , Lisina/química , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/química , Pele/enzimologia , Animais , Colágeno Tipo I/biossíntese , Colágeno Tipo I/genética , Ciclofilinas/genética , Ciclofilinas/ultraestrutura , Retículo Endoplasmático/química , Retículo Endoplasmático/enzimologia , Glicosilação , Heterozigoto , Hidroxilação , Lisina/genética , Espectrometria de Massas , Camundongos , Camundongos Knockout , Microscopia de Força Atômica , Pró-Colágeno-Lisina 2-Oxoglutarato 5-Dioxigenase/genética , Processamento de Proteína Pós-Traducional/genética , Pele/química
7.
Nat Chem ; 11(7): 644-652, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182821

RESUMO

A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.


Assuntos
Lisina/química , Peptídeos Cíclicos/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Ubiquitinas/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Células HeLa , Humanos , Estrutura Molecular , Peptídeos Cíclicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitinas/síntese química , Ubiquitinas/química
9.
BMC Bioinformatics ; 20(1): 346, 2019 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-31208321

RESUMO

BACKGROUND: Acetylation on lysine is a widespread post-translational modification which is reversible and plays a crucial role in some biological activities. To better understand the mechanism, it is necessary to identify acetylation sites in proteins accurately. Computational methods are popular because they are more convenient and faster than experimental methods. In this study, we proposed a new computational method to predict acetylation sites in human by combining sequence features and structural features including physicochemical property (PCP), position specific score matrix (PSSM), auto covariation (AC), residue composition (RC), secondary structure (SS) and accessible surface area (ASA), which can well characterize the information of acetylated lysine sites. Besides, a two-step feature selection was applied, which combined mRMR and IFS. It finally trained a cascade classifier based on SVM, which successfully solved the imbalance between positive samples and negative samples and covered all negative sample information. RESULTS: The performance of this method is measured with a specificity of 72.19% and a sensibility of 76.71% on independent dataset which shows that a cascade SVM classifier outperforms single SVM classifier. CONCLUSIONS: In addition to the analysis of experimental results, we also made a systematic and comprehensive analysis of the acetylation data.


Assuntos
Biologia Computacional/métodos , Máquina de Vetores de Suporte , Acetilação , Sequência de Aminoácidos , Animais , Bases de Dados de Proteínas , Ontologia Genética , Humanos , Lisina/química , Camundongos , Anotação de Sequência Molecular , Matrizes de Pontuação de Posição Específica , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteínas/química , Proteínas/metabolismo , Ratos
10.
Food Chem ; 294: 123-129, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31126444

RESUMO

We previously reported a humidity-mediated method to effectively remove methanol from the crystal lattice of 3',5'-cyclic monophosphate sodium (cAMPNa) methanol trihydrate, converting it to the pentahydrate without changing its inherent orthorhombic packing mode, and preserving its stability. In this paper, we expand this approach to the removal of residual solvents from l-lysine l-glutamate salt and inosine-5'-monophosphate, and contrast the humidity-mediated method with a solvent-mediated method and a conventional drying method. The packing density of the products obtained from the humidity-mediated method were ∼60% higher than those of the products obtained from the solvent-mediated method, and their stability is ∼5-10% higher than those obtained from the solvent-mediated and traditional drying methods. Furthermore, the humidity-mediated method can remove residual methanol more completely. Therefore, the humidity-mediated method can be regarded as a simple and effective route to eliminate residual solvent from crystal lattice for some crystal products, especially residual methanol.


Assuntos
Ácido Glutâmico/química , Lisina/química , Solventes/química , Cristalização , Umidade , Inosina Monofosfato/química , Metanol/química , Difração de Raios X
11.
J Agric Food Chem ; 67(22): 6350-6358, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31083944

RESUMO

α-Dicarbonyls are reactive intermediates formed during Maillard reactions and carbohydrate degradation. The formation of seven α-dicarbonyls was characterized in solutions containing dairy related carbohydrates (galactose, glucose, lactose, and galacto-oligosaccharides (GOS)) during incubations at 40 and 50 °C with and without Nα-acetyl-l-lysine at pH 6.8 for up to 2 months. The concentrations of α-dicarbonyls in samples of monosaccharides with Nα-acetyl-l-lysine were found to be 3-deoxyglucosone (3-DG) > 3-deoxygalactosone (3-DGal) > glyoxal > glucosone, galactosone > methylglyoxal > diacetyl. The presence of Nα-acetyl-l-lysine resulted in up to 100-fold higher concentrations of C6 α-dicarbonyls but lesser formation of glyoxal in the monosaccharide-containing models compared to what was observed in the absence of Nα-acetyl-l-lysine. Galactose incubated with Nα-acetyl-l-lysine generated the highest concentrations of 3-DGal (up to 130 µM), glyoxal (up to 100 µM), and methylglyoxal (up to 9 µM) compared to the other carbohydrates during incubation. Surprisingly, 3-DG (1500 µM) and 3-DGal (80 µM) were formed at levels of 2 orders of magnitude higher in solutions of GOS in the absence of Nα-acetyl-l-lysine as compared to the other carbohydrates at 40 °C, while GOS generated the lowest levels of glyoxal. GOS are widely used as an ingredient in various types of foods products, and it is therefore of importance to consider the risk of generating high levels of the reactive C6 α-dicarbonyl, 3-DG, in these types of products. This study contributes to the understanding of major α-dicarbonyl formation as affected by the presence of primary amines in GOS-, lactose-, and galactose-containing solutions under moderate heating in liquid foods.


Assuntos
Galactose/química , Glucose/química , Glioxal/química , Lactose/química , Lisina/química , Leite/química , Oligossacarídeos/química , Animais , Bovinos , Laticínios/análise , Temperatura Alta , Reação de Maillard , Oxirredução , Aldeído Pirúvico/química
12.
J Agric Food Chem ; 67(23): 6594-6602, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31091091

RESUMO

Modifications of lysine contribute to the amount of dietary advanced glycation end-products reaching the colon. However, little is known about the ability of intestinal bacteria to metabolize dietary N-ε-carboxymethyllysine (CML). Successive transfers of fecal microbiota in growth media containing CML were used to identify and isolate species able to metabolize CML under anaerobic conditions. From our study, only donors exposed to processed foods degraded CML, and anaerobic bacteria enrichments from two of them used 77 and 100% of CML. Oscillibacter and Cloacibacillus evryensis increased in the two donors after the second transfer, highlighting that the bacteria from these taxa could be candidates for anaerobic CML degradation. A tentative identification of CML metabolites produced by a pure culture of Cloacibacillus evryensis was performed by mass spectrometry: carboxymethylated biogenic amines and carboxylic acids were identified as CML degradation products. The study confirmed the ability of intestinal bacteria to metabolize CML under anoxic conditions.


Assuntos
Bactérias/metabolismo , Colo/microbiologia , Microbioma Gastrointestinal , Produtos Finais de Glicação Avançada/metabolismo , Lisina/análogos & derivados , Adulto , Anaerobiose , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Fezes/microbiologia , Produtos Finais de Glicação Avançada/química , Glicosilação , Humanos , Lactente , Lisina/química , Lisina/metabolismo
13.
BMC Genomics ; 20(1): 340, 2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31060518

RESUMO

BACKGROUND: Lysine crotonylation, as a novel evolutionarily conserved type of post-translational modifications, is ubiquitous and essential in cell biology. However, its functions in tea plants are largely unknown, and the full functions of lysine crotonylated proteins of tea plants in nitrogen absorption and assimilation remains unclear. Our study attempts to describe the global profiling of nonhistone lysine crotonylation in tea leaves and to explore how ammonium (NH4+) triggers the response mechanism of lysine crotonylome in tea plants. RESULTS: Here, we performed the global analysis of crotonylome in tea leaves under NH4+ deficiency/resupply using high-resolution LC-MS/MS coupled with highly sensitive immune-antibody. A total of 2288 lysine crotonylation sites on 971 proteins were identified, of which contained in 15 types of crotonylated motifs. Most of crotonylated proteins were located in chloroplast (37%) and cytoplasm (33%). Compared with NH4+ deficiency, 120 and 151 crotonylated proteins were significantly changed at 3 h and 3 days of NH4+ resupply, respectively. Bioinformatics analysis showed that differentially expressed crotonylated proteins participated in diverse biological processes such as photosynthesis (PsbO, PsbP, PsbQ, Pbs27, PsaN, PsaF, FNR and ATPase), carbon fixation (rbcs, rbcl, TK, ALDO, PGK and PRK) and amino acid metabolism (SGAT, GGAT2, SHMT4 and GDC), suggesting that lysine crotonylation played important roles in these processes. Moreover, the protein-protein interaction analysis revealed that the interactions of identified crotonylated proteins diversely involved in photosynthesis, carbon fixation and amino acid metabolism. Interestingly, a large number of enzymes were crotonylated, such as Rubisco, TK, SGAT and GGAT, and their activities and crotonylation levels changed significantly by sensing ammonium, indicating a potential function of crotonylation in the regulation of enzyme activities. CONCLUSIONS: The results indicated that the crotonylated proteins had a profound influence on metabolic process of tea leaves in response to NH4+ deficiency/resupply, which mainly involved in diverse aspects of primary metabolic processes by sensing NH4+, especially in photosynthesis, carbon fixation and amino acid metabolism. The data might serve as important resources for exploring the roles of lysine crotonylation in N metabolism of tea plants. Data were available via ProteomeXchange with identifier PXD011610.


Assuntos
Compostos de Amônio/farmacologia , Camellia sinensis/metabolismo , Crotonatos/química , Lisina/química , Proteínas de Plantas/metabolismo , Processamento de Proteína Pós-Traducional , Proteoma/análise , Camellia sinensis/efeitos dos fármacos , Camellia sinensis/crescimento & desenvolvimento , Biologia Computacional , Fotossíntese , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Mapas de Interação de Proteínas
14.
J Agric Food Chem ; 67(20): 5874-5881, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31050431

RESUMO

Dicarbonyl compounds such as methylglyoxal (MGO) and 3-deoxyglucosone (3-DG) are formed via caramelization and the Maillard reaction in food during heating or in vivo as byproducts of glycolysis. Recently, it was shown that creatine, an amino compound linked to the energy metabolism in vertebrate muscle, reacts rapidly with methylglyoxal under physiological conditions to form N-(4-methyl-5-oxo-1-imidazolin-2-yl)sarcosine (MG-HCr), a methylglyoxal-derived hydroimidazolone of creatine. Based on the observation that heated meat contains only small amounts of MGO and 3-DG when compared to many other foodstuffs, the aim of this study was to investigate a possible reaction of creatine with 3-DG and MGO in meat. From incubation mixtures consisting of 3-DG and creatine, a new hydroimidazolone of creatine, namely N-(4-butyl-1,2,3-triol-5-oxo-1-imidazolin-2-yl)sarcosine (3-DG-HCr), was isolated and characterized via spectroscopic means. To quantitate 3-DG-HCr and MG-HCr, meat and fish products were analyzed via HPLC-MS/MS using isotopically labeled standard material. Whereas samples of raw fish and meat contained only trace amounts of the hydroimidazolones (below 5 µg/kg), up to 28.3 mg/kg MG-HCr and up to 15.3 mg/kg 3-DG-HCr were found in meat and fish products. The concentrations were dependent on the heat treatment and presumably on the smoking process. In comparison to the lysine and arginine derivatives CEL, pyrraline, and MG-H1, the derivatization rate of creatine as MG-HCr and 3-DG-HCr was higher than of lysine and arginine, which clearly demonstrates the 1,2-dicarbonyl scavenging properties of creatine in meat.


Assuntos
Creatina/química , Desoxiglucose/análogos & derivados , Imidazóis/química , Carne/análise , Aldeído Pirúvico/química , Animais , Arginina/química , Bovinos , Galinhas , Cromatografia Líquida de Alta Pressão , Culinária , Desoxiglucose/química , Temperatura Alta , Lisina/química , Reação de Maillard , Suínos , Espectrometria de Massas em Tandem
15.
Nat Cell Biol ; 21(5): 592-602, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962575

RESUMO

Inverted formin 2 (INF2) is a member of the formin family of actin assembly factors. Dominant missense mutations in INF2 are linked to two diseases: focal segmental glomerulosclerosis, a kidney disease, and Charcot-Marie-Tooth disease, a neuropathy. All of the disease mutations map to the autoinhibitory diaphanous inhibitory domain. Interestingly, purified INF2 is not autoinhibited, suggesting the existence of other cellular inhibitors. Here, we purified an INF2 inhibitor from mouse brain tissue, and identified it as a complex of lysine-acetylated actin (KAc-actin) and cyclase-associated protein (CAP). Inhibition of INF2 by CAP-KAc-actin is dependent on the INF2 diaphanous inhibitory domain (DID). Treatment of CAP-KAc-actin-inhibited INF2 with histone deacetylase 6 releases INF2 inhibition, whereas inhibitors of histone deacetylase 6 block the activation of cellular INF2. Disease-associated INF2 mutants are poorly inhibited by CAP-KAc-actin, suggesting that focal segmental glomerulosclerosis and Charcot-Marie-Tooth disease result from reduced CAP-KAc-actin binding. These findings reveal a role for KAc-actin in the regulation of an actin assembly factor by a mechanism that we call facilitated autoinhibition.


Assuntos
Actinas/genética , Proteínas de Transporte/genética , Doença de Charcot-Marie-Tooth/genética , Glomerulosclerose Segmentar e Focal/genética , Proteínas dos Microfilamentos/genética , Actinas/química , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Transporte/química , Doença de Charcot-Marie-Tooth/patologia , Glomerulosclerose Segmentar e Focal/patologia , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/genética , Humanos , Lisina/química , Camundongos , Proteínas dos Microfilamentos/química , Mutação , Ligação Proteica , Domínios Proteicos/genética
16.
Mol Cell ; 74(5): 1010-1019.e6, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-30981630

RESUMO

The essential histone H3 lysine 79 methyltransferase Dot1L regulates transcription and genomic stability and is deregulated in leukemia. The activity of Dot1L is stimulated by mono-ubiquitination of histone H2B on lysine 120 (H2BK120Ub); however, the detailed mechanism is not understood. We report cryo-EM structures of human Dot1L bound to (1) H2BK120Ub and (2) unmodified nucleosome substrates at 3.5 Å and 4.9 Å, respectively. Comparison of both structures, complemented with biochemical experiments, provides critical insights into the mechanism of Dot1L stimulation by H2BK120Ub. Both structures show Dot1L binding to the same extended surface of the histone octamer. In yeast, this surface is used by silencing proteins involved in heterochromatin formation, explaining the mechanism of their competition with Dot1. These results provide a strong foundation for understanding conserved crosstalk between histone modifications found at actively transcribed genes and offer a general model of how ubiquitin might regulate the activity of chromatin enzymes.


Assuntos
Histona-Lisina N-Metiltransferase/química , Histonas/química , Lisina/química , Conformação Proteica , Sítios de Ligação , Microscopia Crioeletrônica , Genoma Humano/genética , Instabilidade Genômica/genética , Heterocromatina/química , Heterocromatina/genética , Histona-Lisina N-Metiltransferase/genética , Histonas/genética , Humanos , Leucemia/genética , Lisina/genética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Nucleossomos/química , Nucleossomos/genética , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Genética , Ubiquitinação/genética
17.
Org Lett ; 21(9): 3265-3270, 2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-30986070

RESUMO

A traceless ß-mercaptan-assisted α-selective ligation of N-terminal lysine-containing peptides has been developed. In this ligation-desulfurization-based protocol, the ε-amine of lysine is free of protection, thus improving the overall synthetic efficiency and avoiding harsh reactions in preparing large peptides and proteins. The applicability of this methodology has been demonstrated in the synthesis of an acid-labile therapeutic protein, interferon gamma, and the anticancer activity of synthetic protein has also been evaluated.


Assuntos
Antineoplásicos/síntese química , Interferon gama/síntese química , Lisina/química , Aminas/química , Antineoplásicos/farmacologia , Sítios de Ligação , Sobrevivência Celular/efeitos dos fármacos , Células HeLa , Humanos , Interferon gama/farmacologia , Conformação Proteica , Técnicas de Síntese em Fase Sólida , Compostos de Sulfidrila/química
18.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(2): 158-161, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30975281

RESUMO

Objective To clarify the interaction between CCAAT enhancer-binding protein α (C/EBPα) and small ubiquitin-related modification (SUMO) in human alveolar type II epithelial cells (AECII), and further identify its modification sites. Methods The expression of C/EBPα and SUMO1 in human AECII was detected by immunofluorescence double labeling. Co-IP was used to detect the interaction of C/EBPα and SUMO1 in AEC II. The SUMO site of C/EBPα was predicted to be the 161st lysine (K161) by the SUMOsp software. The wild-type GFP-C/EBPα plasmids and mutant GFP-K161R plasmids were constructed and transfected into AECII. The SUMO site of C/EBPα was identified by Co-IP. Results Immunofluorescence double staining found that SUMO1 and C/EBPα were co-located in the nucleus. C/EBPα-SUMO band could be marked by Co-IP, which suggested that C/EBPα could interact with SUMO1.When AECII was transfected by wild-type GFP-C/EBPα plasmids. C/EBPα-SUMO1 band could be detected by immunoprecipitation (IP), but could not be detected when transfected by mutant GFP-C/EBPα plasmids. These suggested that the SUMO site of C/EBPα was the 161st lysine. Conclusion C/EBPα can be modified by SUMO1 and the site of its modification is the 161st lysine in human AECII.


Assuntos
Células Epiteliais Alveolares , Proteína alfa Estimuladora de Ligação a CCAAT , Lisina , Proteína SUMO-1 , Proteína alfa Estimuladora de Ligação a CCAAT/química , Humanos , Lisina/química , Lisina/metabolismo , Proteína SUMO-1/metabolismo
19.
Anal Bioanal Chem ; 411(18): 4159-4166, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30993368

RESUMO

Because of structural flexibility and acid lability, the identification of phosphorylated lysine (pLys) peptides is a great challenge. We report here a cleavable hydrophobic derivatization (CHD) strategy for the enrichment and identification of pLys peptides. First, 2,5-dioxopyrrolidin-1-yl-3-(decyldisulfanyl)propanoate was synthesized to react with dephosphorylated lysine peptides, and then the derived peptides were captured by a C18 column, followed by cleavage of the hydrophobic chain, with the specific label left on the target peptides for further identification. By CHD, the enrichment of pLys peptides from interfering peptides (1:1000 mass ratio) was achieved. Furthermore, CHD was applied to screen the pLys targets from Escherichia coli lysates, and 39 pLys sites from 35 proteins were identified. Gene Ontology (GO) analysis showed that these proteins played vital roles in catabolism, metabolism, biogenesis, and biosynthetic processes. All these results demonstrate that CHD might pave the way for comprehensive profiling of the pLys proteome.


Assuntos
Lisina/química , Peptídeos/química , Cromatografia Líquida/métodos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Fosforilação , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas em Tandem/métodos
20.
Molecules ; 24(7)2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30935092

RESUMO

Furosine (Nε-(2-furoylmethyl)-l-lysine) is formed during the early stages of the Maillard reaction from a lysine Amadori compound and is frequently used as a marker of reaction progress. Furosine is toxic, with significant effects on animal livers, kidneys, and other organs. However, reports on the formation of furosine in processed velvet antler are scarce. In this study, we have quantified the furosine content in processed velvet antler by using UPLC-MS/MS. The furosine contents of velvet antler after freeze-drying, boiling, and processing without and with blood were 148.51⁻193.93, 168.10⁻241.22, 60.29⁻80.33, and 115.18⁻138.99 mg/kg protein, respectively. The factors affecting furosine formation in processed velvet antler, including reducing sugars, proteins, amino acids, and process temperature, are discussed herein. Proteins, amino acids, and reducing sugars are substrates for the Maillard reaction and most significantly influence the furosine content in the processed velvet antler. High temperatures induce the production of furosine in boiled velvet antler but not in the freeze-dried samples, whereas more furosine is produced in velvet antler processed with blood, which is rich in proteins, amino acids, and reducing sugars, than in the samples processed without blood. Finally, wax slices rich in proteins, amino acids, and reducing sugars produced more furosine than the other parts of the velvet antler. These data provide a reference for guiding the production of low-furosine velvet antler and can be used to estimate the consumer intake of furosine from processed velvet antler.


Assuntos
Chifres de Veado/química , Lisina/análogos & derivados , Aminoácidos/química , Animais , Cromatografia Líquida , Lisina/química , Reação de Maillard , Estrutura Molecular , Reprodutibilidade dos Testes , Açúcares/química , Espectrometria de Massas em Tandem
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