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1.
Food Chem ; 304: 125448, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31491713

RESUMO

Blood, from slaughterhouses, is an inevitable part of meat production, causing environmental problems due to the large volumes recovered and its low valorization. However, the α137-141 peptide, a natural antimicrobial peptide, can be obtained after hydrolysis of hemoglobin, the main constituent of blood red part. To recover it at a sufficient concentration for antimicrobial applications, a new sustainable technology, called electrodialysis with ultrafiltration membrane (EDUF), was investigated. The α137-141 concentration was increased about 4-fold at a feed peptide concentration of 8% with an enrichment factor above 24-fold. This feed peptide concentration also needed the lowest relative energy consumption. Moreover, this peptide fraction protected meat against microbial growth, as well as rancidity, during 14 days under refrigeration. This peptide fraction was validated as a natural preservative and substitute for synthetic additives against food spoilage. Finally, producing antimicrobial/antioxidant peptide from wastes by EDUF fits perfectly with the concept of circular economy.

2.
Neurobiol Dis ; 129: 217-233, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30928644

RESUMO

Alzheimer's Disease is a devastating dementing disease involving amyloid deposits, neurofibrillary tangles, progressive and irreversible cognitive impairment. Today, only symptomatic drugs are available and therapeutic treatments, possibly acting at a multiscale level, are thus urgently needed. To that purpose, we designed multi-effects compounds by synthesizing drug candidates derived by substituting a novel N,N'-disubstituted piperazine anti-amyloid scaffold and adding acetylcholinesterase inhibition property. Two compounds were synthesized and evaluated. The most promising hybrid molecule reduces both the amyloid pathology and the Tau pathology as well as the memory impairments in a preclinical model of Alzheimer's disease. In vitro also, the compound reduces the phosphorylation of Tau and inhibits the release of Aß peptides while preserving the processing of other metabolites of the amyloid precursor protein. We synthetized and tested the first drug capable of ameliorating both the amyloid and Tau pathology in animal models of AD as well as preventing the major brain lesions and associated memory impairments. This work paves the way for future compound medicines against both Alzheimer's-related brain lesions development and the associated cognitive impairments.

3.
Artigo em Inglês | MEDLINE | ID: mdl-29692758

RESUMO

A qualitative study is presented, where the main question was whether food-derived hemorphins, i.e., originating from digested alimentary hemoglobin, could pass the intestinal barrier and/or the blood-brain barrier (BBB). Once absorbed, hemorphins are opioid receptor (OR) ligands that may interact with peripheral and central OR and have effects on food intake and energy balance regulation. LLVV-YPWT (LLVV-H4), LVV-H4, VV-H4, VV-YPWTQRF (VV-H7), and VV-H7 hemorphins that were previously identified in the 120 min digest resulting from the simulated gastrointestinal digestion of hemoglobin have been synthesized to be tested in in vitro models of passage of IB and BBB. LC-MS/MS analyses yielded that all hemorphins, except the LLVV-H4 sequence, were able to cross intact the human intestinal epithelium model with Caco-2 cells within 5-60 min when applied at 5 mM. Moreover, all hemorphins crossed intact the human BBB model with brain-like endothelial cells (BLEC) within 30 min when applied at 100 µM. Fragments of these hemorphins were also detected, especially the YPWT common tetrapeptide that retains OR-binding capacity. A cAMP assay performed in Caco-2 cells indicates that tested hemorphins behave as OR agonists in these cells by reducing cAMP production. We further provide preliminary results regarding the effects of hemorphins on tight junction proteins, specifically here the claudin-4 that is involved in paracellular permeability. All hemorphins at 100 µM, except the LLVV-H4 peptide, significantly decreased claudin-4 mRNA levels in the Caco-2 intestinal model. This in vitro study is a first step toward demonstrating food-derived hemorphins bioavailability which is in line with the growing body of evidence supporting physiological functions for food-derived peptides.

4.
Nitric Oxide ; 71: 32-43, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-29051112

RESUMO

PURPOSE: In a previous work, we have synthetized a new dinitrosothiol, i.e. S,S'-dinitrosobucillamine BUC(NO)2 combining S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO) in its structure. When exposed to isolated aorta, we observed a 1.5-fold increase of •NO content and a more potent vasorelaxation (1 log higher pD2) compared to NACNO and SNAP alone or combined (Dahboul et al., 2014). In the present study, we analyzed the thermodynamics and kinetics for the release of •NO through computational modeling techniques and correlated it to plasma assays. Then BUC(NO)2 was administered in vivo to rats, assuming it will induce higher and/or longer hypotensive effects than its two constitutive S-mononitrosothiols. METHODS: Free energies for the release of •NO entities have been computed at the density functional theory level assuming an implicit model for the aqueous environment. Degradation products of BUC(NO)2 were evaluated in vitro under heating and oxidizing conditions using HPLC coupled with tandem mass spectrometry (MS/MS). Plasma from rats were spiked with RSNO and kinetics of RSNO degradation was measured using the classical Griess-Saville method. Blood pressure was measured in awake male Wistar rats using telemetry (n = 5, each as its own control, 48 h wash-out periods between subcutaneous injections under transient isoflurane anesthesia, random order: 7 mL/kg vehicle, 3.5, 7, 14 µmol/kg SNAP, NACNO, BUC(NO)2 and an equimolar mixture of SNAP + NACNO in order to mimic the number of •NO contained in BUC(NO)2). Variations of mean (ΔMAP, reflecting arterial dilation) and pulse arterial pressures (ΔPAP, indirectly reflecting venodilation, used to determine effect duration) vs. baseline were recorded for 4 h. RESULTS: Computational modeling highlights the fact that the release of the first •NO radical in BUC(NO)2 requires a free energy which is intermediate between the values obtained for SNAP and NACNO. However, the release of the second •NO radical is significantly favored by the concerted formation of an intramolecular disulfide bond. The corresponding oxidized compound was also characterized as related substance obtained under degradation conditions. The in vitro degradation rate of BUC(NO)2 was significantly greater than for the other RSNO. For equivalent low and medium •NO-load, BUC(NO)2 produced a hypotension identical to NACNO, SNAP and the equimolar mixture of SNAP + NACNO, but its effect was greater at higher doses (-62 ± 8 and -47 ± 14 mmHg, maximum ΔMAP for BUC(NO)2 and SNAP + NACNO, respectively). Its duration of effect on PAP (-50%) lasted from 35 to 95 min, i.e. shorter than for the other RSNO (from 90 to 135 min for the mixture SNAP + NACNO). CONCLUSION: A faster metabolism explains the abilities of BUC(NO)2 to release higher amounts of •NO and to induce larger hypotension but shorter-lasting effects than those induced by the SNAP + NACNO mixture, despite an equivalent •NO-load.


Assuntos
Anti-Hipertensivos/uso terapêutico , Cisteína/análogos & derivados , Hipertensão/tratamento farmacológico , Doadores de Óxido Nítrico/uso terapêutico , Compostos Nitrosos/uso terapêutico , Acetilcisteína/análogos & derivados , Acetilcisteína/metabolismo , Acetilcisteína/uso terapêutico , Animais , Anti-Hipertensivos/sangue , Anti-Hipertensivos/química , Anti-Hipertensivos/metabolismo , Pressão Arterial/efeitos dos fármacos , Simulação por Computador , Cisteína/sangue , Cisteína/química , Cisteína/metabolismo , Cisteína/uso terapêutico , Cinética , Masculino , Modelos Químicos , Doadores de Óxido Nítrico/sangue , Doadores de Óxido Nítrico/química , Doadores de Óxido Nítrico/metabolismo , Compostos Nitrosos/sangue , Compostos Nitrosos/química , Compostos Nitrosos/metabolismo , Ratos Wistar , S-Nitroso-N-Acetilpenicilamina/metabolismo , S-Nitroso-N-Acetilpenicilamina/uso terapêutico
5.
J Clin Endocrinol Metab ; 102(10): 3783-3794, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28938455

RESUMO

Context: Bile acids (BAs) are signaling molecules controlling energy homeostasis that can be both toxic and protective for the liver. BA alterations have been reported in obesity, insulin resistance (IR), and nonalcoholic steatohepatitis (NASH). However, whether BA alterations contribute to NASH independently of the metabolic status is unclear. Objective: To assess BA alterations associated with NASH independently of body mass index and IR. Design and Setting: Patients visiting the obesity clinic of the Antwerp University Hospital (a tertiary referral facility) were recruited from 2006 to 2014. Patients: Obese patients with biopsy-proven NASH (n = 32) and healthy livers (n = 26) were matched on body mass index and homeostasis model assessment of IR. Main Outcome Measures: Transcriptomic analyses were performed on liver biopsies. Plasma concentrations of 21 BA species and 7α-hydroxy-4-cholesten-3-one, a marker of BA synthesis, were determined by liquid chromatography-tandem mass spectrometry. Plasma fibroblast growth factor 19 was measured by enzyme-linked immunosorbent assay. Results: Plasma BA concentrations did not correlate with any hepatic lesions, whereas, as previously reported, primary BA strongly correlated with IR. Transcriptomic analyses showed unaltered hepatic BA metabolism in NASH patients. In line, plasma 7α-hydroxy-4-cholesten-3-one was unchanged in NASH. Moreover, no sign of hepatic BA accumulation or activation of BA receptors-farnesoid X, pregnane X, and vitamin D receptors-was found. Finally, plasma fibroblast growth factor 19, secondary-to-primary BA, and free-to-conjugated BA ratios were similar, suggesting unaltered intestinal BA metabolism and signaling. Conclusions: In obese patients, BA alterations are related to the metabolic phenotype associated with NASH, especially IR, but not liver necroinflammation.


Assuntos
Ácidos e Sais Biliares/metabolismo , Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Adulto , Estudos de Casos e Controles , Feminino , Regulação da Expressão Gênica , Humanos , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Obesidade/complicações , Obesidade/epidemiologia
6.
Chemistry ; 23(45): 10777-10788, 2017 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-28488394

RESUMO

In the present study, we report the first silver-dependent enantiodivergent gold-catalysed reaction. The asymmetric intramolecular hydroamination of alkenes catalysed by the combination of a single chiral binuclear gold(I) chloride complex and silver perchlorate can afford both enantiomers of the products by a simple solvent change from toluene to methanol. Such an enantiodivergent reaction is strictly independent of the reaction temperature or of the nature of the catalyst anion and displays the same first-order kinetic rate law with respect to substrate concentration in both solvents. Beyond a simple solvent effect the enantioinversion is controlled by gold-silver chloride adducts which occur only in methanol and allow a dual activation of the reagent. While one single gold atom activates the alkene moiety, the other gold atom forms an oxophilic gold-silver chloride adduct which is likely to interact with the carbamate function. By comparison with toluene, which affords (S)-enantiomer, this proximal and bimetallic activation would allow an opposite stereodifferentiation of the two diastereomeric intermediates during the final protodeauration step and lead therefore to the (R)-enantiomer.

7.
Food Res Int ; 89(Pt 1): 382-390, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28460928

RESUMO

Dietary proteins have been reported to induce a strong feeling of satiety that has been partially explained by gut hormone level increase. Up to date, various protein hydrolysates have demonstrated in vitro and in vivo their potential to stimulate gut hormone secretion related to food intake decrease and their mechanisms of action have just started to be resolved. In this context, this study aimed at identifying new peptide sequences involved in gut hormone secretion released by protein in vitro gastrointestinal digestion. Targeted gut hormones were Cholecystokinin (CCK) and Glucagon-Like Peptide 1 (GLP-1). The activity of DPP-IV was also considered as it strongly modulates GLP-1 action. In a previous study, simulated digestion of dietary protein has generated hydrolysates with enhancing effect on CCK and GLP-1 secretion in STC-1 cells as well as DPP-IV inhibitory properties. Successive purification steps were performed to isolate peptide fractions involved in these bioactivities whose sequence was determined by LC-MS-MS. Three peptide sequences ANVST, TKAVEH and KAAVT were pointed out for their stimulating effects on GLP-1 secretion. The sequence VAAA was isolated for its DPP-IV inhibitory properties. Two peptide groups were strongly involved in CCK release sharing a certain occurrence of aromatic amino acid residues.

8.
Cell Metab ; 22(3): 418-26, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26235421

RESUMO

The interest in brown adipose tissue (BAT) as a target to combat metabolic disease has recently been renewed with the discovery of functional BAT in humans. In rodents, BAT can be activated by bile acids, which activate type 2 iodothyronine deiodinase (D2) in BAT via the G-coupled protein receptor TGR5, resulting in increased oxygen consumption and energy expenditure. Here we examined the effects of oral supplementation of the bile acid chenodeoxycholic acid (CDCA) on human BAT activity. Treatment of 12 healthy female subjects with CDCA for 2 days resulted in increased BAT activity. Whole-body energy expenditure was also increased upon CDCA treatment. In vitro treatment of primary human brown adipocytes derived with CDCA or specific TGR5 agonists increased mitochondrial uncoupling and D2 expression, an effect that was absent in human primary white adipocytes. These findings identify bile acids as a target to activate BAT in humans.


Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Tecido Adiposo Marrom/metabolismo , Ácido Quenodesoxicólico/farmacologia , Metabolismo Energético/efeitos dos fármacos , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/metabolismo , Administração Oral , Adulto , Células Cultivadas , Ácido Quenodesoxicólico/administração & dosagem , Ácido Quenodesoxicólico/sangue , Feminino , Humanos , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Adulto Jovem
9.
Probiotics Antimicrob Proteins ; 5(3): 176-86, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26782986

RESUMO

The use of antimicrobial peptides (AMPs) is an alternative to traditional antibiotics. AMPs are obtained using different methods such as bacterial synthesis, chemical synthesis and controlled enzymatic hydrolysis. The later is an interesting approach that deserves our attention because of the yields gathered and peptides engineered. Usually, activities of AMPs obtained in such a way are tightly dependent on the hydrolysis mechanism used. This paper deals with the hydrolysis of hemoglobin mechanism as a potential source of AMPs. Production of AMPs from hemoglobin using enzymatic controlled system is linked to hemoglobin structure. Further, we show that bovine hemoglobin, which is sensitive to peptic hydrolysis, results upon enzymatic digestion as a great source of AMPs. The hemoglobin in native and denatured states was hydrolyzed by "one-by-one" and "zipper" mechanisms, respectively. Nevertheless, a new mechanism named "semi-zipper" mechanism is obtained when protein is in molten globule structural state, constituting an original strategy for AMPs production. Seventy seven percentage of the peptides obtained by this new strategy showed antibacterial activity against nine strains.

10.
Int J Biol Macromol ; 49(2): 143-53, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21510973

RESUMO

Under standard conditions, the peptides and specially the active peptides were obtained from either the denatured hemoglobin that all structures are completely modified or either the native hemoglobin where all structures are intact. In these conditions, antibacterial peptides were isolated from a very complex peptidic hydrolysate which contains more than one hundred peptides having various sizes and characteristics, involving a complex purification process. The new hydrolysis conditions were obtained by using 40% methanol, 30% ethanol, 20% propanol or 10% butanol. These conditions, where only the secondary structure of hemoglobin retains intact, were followed in order to enrich the hydrolyzed hemoglobin by active peptides or obtain new antibacterial peptides. In these controlled peptic hydrolysis of hemoglobin, a selective and restrictive hydrolysate contained only 29 peptides was obtained. 26 peptides have an antibacterial activity against Micrococcus luteus, Listeria innocua, and Escherichia coli with MIC from 187.1 to 1 µM. Among these peptides, 13 new antibacterial peptides are obtained only in these new hydrolysis conditions.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Hemoglobinas/química , Sequência de Aminoácidos , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/farmacologia , Bactérias/efeitos dos fármacos , Bovinos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Pepsina A/metabolismo
11.
Peptides ; 29(6): 969-77, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18342399

RESUMO

A peptic hemoglobin hydrolysate was fractioned by a semi-preparative reversed-phase HPLC and some fractions have an antibacterial activity against four bacteria strains: Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis. These fractions were analyzed by ESI/MS and ESI/MS/MS, in order to characterize the peptides in these fractions. Each fraction contains at least three peptides and some fractions contain five peptides. All these fractions were purified several times by HPLC to obtain pure peptides. Thirty antibacterial peptides were identified. From the isolated antibacterial peptides, 24 peptides were derived from the alpha chains of hemoglobin and 6 peptides were derived from the beta chains of hemoglobin. The lowest concentration of these peptides (minimum inhibitory concentration (MIC)) necessary to completely inhibit the growth of four bacteria strain was determined. The cell population of all of the tested bacteria species decreased by at least 97% after a 24-h incubation with any of the peptides at the minimum inhibitory concentration.


Assuntos
Antibacterianos/farmacologia , Hemoglobinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/química , Bovinos , Cromatografia Líquida de Alta Pressão , Escherichia coli/efeitos dos fármacos , Hemoglobinas/química , Concentração de Íons de Hidrogênio , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Listeria/efeitos dos fármacos , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Micrococcus luteus/efeitos dos fármacos , Dados de Sequência Molecular , Pepsina A/farmacologia , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Peptídeos/química , Peptídeos/farmacologia , Estrutura Secundária de Proteína , Salmonella enteritidis/efeitos dos fármacos
13.
Peptides ; 27(9): 2082-9, 2006 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16730859

RESUMO

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields several intermediate peptide fractions after separation by reversed phase HPLC exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli, and Salmonella enteritidis. From these fractions, four new antibacterial peptides were isolated and analyzed by ESI-MS/MS. Three of these peptides correspond to fragments of the alpha-chain of bovine hemoglobin: alpha107-141, alpha137-141, and alpha133-141, and one peptide to the beta-chain: beta126-145. The minimum inhibitory concentrations (MIC) of these peptides towards the four strains and their hemolytic activity towards bovine erythrocytes were determined.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Hemoglobinas/química , Hemoglobinas/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antibacterianos/isolamento & purificação , Bovinos , Endorfinas/química , Endorfinas/isolamento & purificação , Endorfinas/farmacologia , Hemoglobinas/isolamento & purificação , Hidrólise , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Fragmentos de Peptídeos/isolamento & purificação
14.
FEBS Lett ; 579(20): 4309-16, 2005 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-16051222

RESUMO

IGF-I is degraded within the endosomal apparatus as a consequence of receptor-mediated endocytosis. However, the nature of the responsible protease and the position of the cleavage sites in the IGF-I molecule remain undefined. In vitro proteolysis of IGF-I using an endosomal lysate required an acidic pH and was sensitive to CA074, an inhibitor of the cathepsin B enzyme. By nondenaturing immunoprecipitation, the acidic IGF-I-degrading activity was attributed to the luminal species of endosomal cathepsin B with apparent molecular masses of 32- and 28-kDa. The cathepsin B precursor, procathepsin B, was processed in vitro within isolated endosomes at pH 5 or at 7 in the presence of ATP, the substrate of the vacuolar H(+)-ATPase. The rate of IGF-I hydrolysis using an endosomal lysate or pure cathepsin B was found to be optimal at pH 5-6 and moderate at pH 4 and 7. Competition studies revealed that EGF and IGF-I share a common binding site on the cathepsin B enzyme, with native IGF-I displaying the lowest affinity for the protease (IC50 approximately 1.5 microM). Hydrolysates of IGF-I generated at low pH by endosomal IGF-I-degrading activity and analyzed by reverse-phase HPLC and mass spectrometry revealed cleavage sites at Lys68-Ser69, Ala67-Lys68, Pro66-Ala67 and Lys65-Pro66 within the C-terminal D-domain of IGF-I. Treatment of human HepG2 hepatoma cells with the cathepsin B proinhibitor CA074-Me reduced, in vivo, the intracellular degradation of internalized [125I]IGF-I and, in vitro, the degradation of exogenous [125I]IGF-I incubated with the cell-lysates at pH 5. Inhibitors of cathepsin B and pro-cathepsin B processing, which abolish endosomal proteolysis of IGF-I and alter tumor cell growth and IGF-I receptor signalling, merit investigation as antimetastatic drugs.


Assuntos
Catepsina B/metabolismo , Endossomos/enzimologia , Fator de Crescimento Insulin-Like I/metabolismo , Catálise , Catepsina B/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Dipeptídeos/farmacologia , Humanos , Fator de Crescimento Insulin-Like I/química , Leucina/análogos & derivados , Leucina/farmacologia , Estrutura Terciária de Proteína , Células Tumorais Cultivadas
15.
Peptides ; 26(5): 713-9, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15808900

RESUMO

Peptic digestion of bovine hemoglobin at low degree of hydrolysis yields an intermediate peptide fraction exhibiting antibacterial activity against Micrococcus luteus A270, Listeria innocua, Escherichia coli and Salmonella enteritidis after separation by reversed-phase HPLC. From this fraction a pure peptide was isolated and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). This peptide correspond to the 107-136 fragment of the alpha chain of bovine hemoglobin. The minimum inhibitory concentrations (MIC) towards the four strains and its hemolytic activity towards bovine erythrocytes were determined. A MIC of 38 microM was reported against L. innocua and 76 microM for other various bacterial species. This peptide had no hemolytic activity up to 380 microM concentration.


Assuntos
Antibacterianos/farmacologia , Hemoglobinas/farmacologia , Fragmentos de Peptídeos/farmacologia , Animais , Antibacterianos/química , Bovinos , Escherichia coli/efeitos dos fármacos , Hemoglobinas/química , Hemólise , Hidrólise , Listeria/efeitos dos fármacos , Micrococcus luteus/efeitos dos fármacos , Fragmentos de Peptídeos/química , Peptídeos/química , Peptídeos/farmacologia , Salmonella enteritidis/efeitos dos fármacos
16.
Biochem J ; 383(Pt. 3): 573-80, 2004 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-15233628

RESUMO

HNF4alpha (hepatocyte nuclear factor 4alpha) belongs to a complex transcription factor network that is crucial for the function of hepatocytes and pancreatic beta-cells. In these cells, it activates the expression of a very large number of genes, including genes involved in the transport and metabolism of glucose and lipids. Mutations in the HNF4alpha gene correlate with MODY1 (maturity-onset diabetes of the young 1), a form of type II diabetes characterized by an impaired glucose-induced insulin secretion. The MODY1 G115S (Gly115-->Ser) HNF4alpha mutation is located in the DNA-binding domain of this nuclear receptor. We show here that the G115S mutation failed to affect HNF4alpha-mediated transcription on apolipoprotein promoters in HepG2 cells. Conversely, in pancreatic beta-cell lines, this mutation resulted in strong impairments of HNF4alpha transcriptional activity on the promoters of LPK (liver pyruvate kinase) and HNF1alpha, with this transcription factor playing a key role in endocrine pancreas. We show as well that the G115S mutation creates a PKA (protein kinase A) phosphorylation site, and that PKA-mediated phosphorylation results in a decreased transcriptional activity of the mutant. Moreover, the G115E (Gly115-->Glu) mutation mimicking phosphorylation reduced HNF4alpha DNA-binding and transcriptional activities. Our results may account for the 100% penetrance of diabetes in human carriers of this mutation. In addition, they suggest that introduction of a phosphorylation site in the DNA-binding domain may represent a new mechanism by which a MODY1 mutation leads to loss of HNF4alpha function.


Assuntos
Idade de Início , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/genética , Diabetes Mellitus/genética , Glicina/genética , Mutação de Sentido Incorreto/genética , Fosfoproteínas/genética , Serina/genética , Fatores de Transcrição/genética , Substituição de Aminoácidos/genética , Sítios de Ligação/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proteína Quinase Tipo II Dependente de AMP Cíclico , DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/fisiologia , Células HeLa/química , Células HeLa/metabolismo , Células HeLa/patologia , Fator 4 Nuclear de Hepatócito , Histidina/biossíntese , Histidina/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Fosfoproteínas/biossíntese , Fosfoproteínas/fisiologia , Fosforilação , Serina/metabolismo , Especificidade por Substrato , Fatores de Transcrição/biossíntese , Fatores de Transcrição/fisiologia
17.
Mol Reprod Dev ; 68(2): 232-9, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15095345

RESUMO

In this article we study the proteins responsible for chromatin condensation during spermiogenesis in the cephalopod Octopus vulgaris. The DNA of ripe sperm nuclei in this species is condensed by a set of five different proteins. Four of these proteins are protamines. The main protamine (Po2), a protein of 44 amino acid residues, is extraordinarily simple (composed of only three different amino acid types: arginine (R), serine (S), and glycine (G). It is a basic molecule consisting of 79.5 mol% arginine residues. The rest of the protamines (Po3, Po4, Po5) are smaller molecules (33, 28, and 30 amino acid residues, respectively) that are homologous among themselves and probably with the main Po2 protamine. The ripe sperm nucleus of O. vulgaris also contains a small quantity of a molecule (Po1) that is similar to Po2 protamine. This protein could represent a Po2 protamine-precursor in a very advanced step of its processing. We discuss the characteristics of these proteins, as well as the relation between the complexity of chromatin condensation and the transitions of nuclear proteins during spermiogenesis in O. vulgaris.


Assuntos
Cromatina/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Octopodiformes/fisiologia , Espermatogênese/fisiologia , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Protaminas/metabolismo
18.
Bioorg Med Chem ; 12(1): 23-9, 2004 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-14697766

RESUMO

The benzo[b]acronycine derivative S23906-1 has been recently identified as a promising antitumor agent, showing remarkable in vivo activities against a panel of solid tumors. The anticancer activity is attributed to the capacity of the drug to alkylate DNA, selectively at the exocyclic 2-amino group of guanine residues. Hydrolysis of the C-1 and C-2 acetate groups of S23906-1 provides the diol compound S28907-1 which is inactive whereas the intermediate C-2 monoacetate derivative S28687-1 is both highly reactive toward DNA and cytotoxic. The reactivity of this later compound S28687-1 toward two bionucleophiles, DNA and the tripeptide glutathion, has been investigated by mass spectrometry to identify the nature of the (type II) covalent adducts characterized by the loss of the acetate group at position 2. On the basis of NMR and molecular modeling analyses, the reaction mechanism is explained by a transesterification process where the acetate leaving group is transferred from position C-2 to C-1. Altogether, the study validates the reaction scheme of benzo[b]acronycine derivative with its target.


Assuntos
Acronina/metabolismo , Antineoplásicos Fitogênicos/metabolismo , DNA/metabolismo , Glutationa/metabolismo , Acronina/análise , Acronina/química , Antineoplásicos Fitogênicos/análise , Antineoplásicos Fitogênicos/química , Sítios de Ligação , DNA/análise , Glutationa/análise , Ligação Proteica/fisiologia , Espectrometria de Massas por Ionização por Electrospray/métodos
19.
Endocrinology ; 144(12): 5308-21, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12970169

RESUMO

Proinsulin, the insulin precursor in pancreatic beta-cells, displays a slower hepatic clearance than insulin and exerts a more prolonged metabolic effect on liver in vivo. To elucidate the mechanisms underlying these differences, the cellular itinerary and processing of proinsulin and insulin in rat liver have been comparatively studied using cell fractionation. As [125I]-insulin, [125I]-proinsulin taken up into liver in vivo was internalized and accumulated in endosomes, in which it underwent dissociation from the insulin receptor and degradation in a pH- and ATP-dependent manner. However, relative to [125I]-insulin, [125I]-proinsulin showed a delayed and prolonged in vivo association with endosomes, a slower in vivo and cell-free endosomal processing, and a higher cell-free endosome-lysosome transfer. Endosomal extracts degraded to a lesser extent proinsulin than insulin at acidic pH; so did, and even proportionally less, at neutral pH, plasma membrane and cytosolic fractions. Proinsulin degradation products generated by soluble endosomal extracts were isolated by HPLC and characterized by mass spectrometry. Under conditions resulting in multiple cleavages in insulin, proinsulin was cleaved at eight bonds in the C peptide but only at the Phe24-Phe25 bond in the insulin moiety. As native insulin, native proinsulin induced a dose- and time-dependent endocytosis and tyrosine phosphorylation of the insulin receptor; but at an inframaximal dose, proinsulin effects on these processes were of longer duration. We conclude that a reduced proteolysis of proinsulin in endosomes, and probably also at the plasma membrane, accounts for its slower hepatic clearance and prolonged effects on insulin receptor endocytosis and tyrosine phosphorylation.


Assuntos
Fígado/metabolismo , Proinsulina/farmacocinética , Animais , Sistema Livre de Células , Endocitose/fisiologia , Endossomos/metabolismo , Hipoglicemiantes/farmacocinética , Técnicas In Vitro , Insulina/farmacocinética , Radioisótopos do Iodo , Lisossomos/metabolismo , Masculino , Fosforilação , Ratos , Ratos Sprague-Dawley , Receptor de Insulina/metabolismo , Frações Subcelulares/metabolismo , Tirosina/metabolismo
20.
Endocrinology ; 144(12): 5353-64, 2003 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12959981

RESUMO

We have investigated the proteolytic mechanisms of glucagon degradation within hepatic endosomes at neutral pH before lumen acidification. Hepatic endosomes incubated at neutral pH rapidly degraded native glucagon into 13 intermediate products, one of which corresponded to the bioactive fragment glucagon-(19-29) (miniglucagon). The serine protease inhibitor phenylmethylsulfonyl fluoride as well as the nonspecific protease inhibitor bacitracin inhibited the endosomal degradation of glucagon at pH 7. In purified endosomal fractions, miniglucagon endopeptidase was undetectable as evaluated by immunoblotting, and immunoprecipitation with antibodies to insulin-degrading enzyme, cathepsins B and D, or furin failed to remove the endosomal neutral glucagonase activity. Incubation of endosomal fractions and [125I]iodoglucagon with the zero-length bifunctional cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide resulted in specific labeling of a 170-kDa polypeptide. The labeling was completely inhibited by unlabeled glucagon (IC50 value, 5 x 10-7 m) and bacitracin (IC50 value, 1 microg/ml), suggesting that it may correspond to a bacitracin-sensitive glucagon-degrading enzyme. Treatment of the 125I-labeled 170-kDa cross-linked polypeptide with N-glycanase demonstrated that the cross-linked complex contained approximately 30 kDa of N-linked oligosaccharides. Specific cross-linking of the 170-kDa polypeptide was also observed using [125I]Tyr12-miniglucagon as the radioligand. Together, these data suggest that the 170-kDa glycoprotein represents a novel glucagon-degrading activity that could mediate glucagon proteolysis within endosomes before the acidification step and generate the bioactive (19-29) miniglucagon peptide.


Assuntos
Endossomos/enzimologia , Glucagon/farmacocinética , Concentração de Íons de Hidrogênio , Fragmentos de Peptídeos/farmacocinética , Peptídeo Hidrolases/metabolismo , Marcadores de Afinidade , Animais , Bacitracina/farmacologia , Furina , Insulisina , Radioisótopos do Iodo , Masculino , Mitocôndrias/metabolismo , Inibidores de Proteases/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Glucagon , Especificidade por Substrato
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