RESUMO
BACKGROUND: Skeletal muscle synthesizes, stores, and releases body L-glutamine (GLN). Muscle atrophy due to disabling diseases triggers the activation of proteolytic and pro-apoptotic cell signaling, thus impairing the body's capacity to manage GLN content. This situation has a poor therapeutic prognosis. OBJECTIVE: Evaluating if oral GLN supplementation can attenuate muscle wasting mediated by elevated plasma cortisol and activation of caspase-3, p38MAPK, and FOXO3a signaling pathways in soleus and gastrocnemius muscles of rats submitted to 14-day bilateral hindlimbs immobilization. METHODS: Animals were randomly distributed into six groups: non-immobilized rats (Control), control orally supplemented with GLN (1 g kg-1) in solution with L-alanine (ALA: 0.61 g kg-1; GLN+ALA), control orally supplemented with dipeptide L-alanyl-L-glutamine (DIP; 1.49 g kg-1), hindlimbs immobilized rats (IMOB), IMOB orally GLN+ALA supplemented (GLN+ALA-IMOB), and IMOB orally DIP supplemented (DIP-IMOB). Plasma and muscle GLN concentration, plasma cortisol level, muscle caspase-3 activity, muscle p38MAPK and FOXO3a protein content (total and phosphorylated forms), and muscle cross-sectional area (CSA) were measured. RESULTS: Compared to controls, IMOB rats presented: a) increased plasma cortisol levels; b) decreased plasma and muscle GLN concentration; c) increased muscle caspase-3 activity; d) increased total and phosphorylated p38MAPK protein content; e) increased FOXO3a and decreased phosphorylated FOXO3a protein content; f) reduced muscle weight and CSA befitting to atrophy. Oral supplementation with GLN+ALA and DIP was able to significantly attenuate these effects. CONCLUSIONS: These findings attest that oral GLN supplementation in GLN+ALA solution or DIP forms attenuates rats' skeletal muscle mass wasting caused by disuse-mediated muscle atrophy.
Assuntos
Glutamina , Hidrocortisona , Atrofia Muscular , Animais , Ratos , Caspase 3/metabolismo , Suplementos Nutricionais , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Glutamina/farmacologia , Músculo Esquelético , Atrofia Muscular/tratamento farmacológico , Atrofia Muscular/etiologia , Atrofia Muscular/metabolismo , Transdução de Sinais , Proteína Forkhead Box O3/efeitos dos fármacos , Proteína Forkhead Box O3/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The study aimed to compare the effects of crystalline L-lysine and L-glutamate (CAA), Lys-Glu dipeptide (KE) on the growth and muscle development of grass carp (Ctenopharyngodon idellus), and related molecular mechanisms. Five experimental diets (CR, 0.5% CAA, 1.5% CAA, 0.5% KE, 1.5% KE) containing Lys and Glu as free (Lys and Glu, CAA) dipeptide (Lys-Glu, KE) forms were prepared, respectively. A total of 450 juvenile grass carp with an initial weight of 10.69 ± 0.07 g were randomly assigned to 15 cages, and 5 treatments with 3 replicates of 30 fish each for 61 days of feeding. The results showed that the group of 0.5% KE exhibited the best growth performances according to the indicator's weight gain rate (WGR) and specific growth rate (SGR), although no statistically significant occurred among all groups; diet supplemented with 0.5% CAA significantly elevated the condition factor (CF) and viscerasomatic index (VSI) of juvenile grass carp. Diet supplemented with different Lys and Glu co-forms at different levels promoted the muscle amino acid content compared with those of CR group. Comparing with the CR group and other groups, the hardness of 0.5% CAA group significantly increased, and the springiness of 0.5% KE group excelled. Both the muscle fiber diameter and density of 0.5% KE group showed significant difference with those of the CR group, and a negative correlation between them was also observed. To uncover the related molecular mechanism of the differences caused by the different co-forms of Lys and Glu, the effect of different diets on the expressions of protein absorption, muscle quality, and antioxidation-related genes was analyzed. The results suggested that comparing with those of CR group, the dipeptide KE inhibited the expressions of genes associated with protein metabolism, such as AKT, S6K1, and FoxO1a but promoted PCNA expression, while the free style of CAA would improve the FoxO1a expression. Additionally, the muscle development-related genes (MyoD, MyOG, and Myf5) were significantly boosted in CAA co-form groups, and the expressions of fMYHCs were blocked but fMYHCs30 significantly promoted in 0.5% KE group. Finally, the effect of different co-forms of Lys and Glu on muscle antioxidant was examined. The 0.5% CAA diet was verified to increase GPX1a but obstruct Keap1 and GSTP1 expressions, resulting in enhanced SOD activity and reduced MDA levels in plasma. Collectively, the different co-forms of Lys and Glu influenced the growth of juvenile grass carp, and also the muscle development and quality through their different regulation on the protein metabolism, muscle development- and antioxidative-related genes.
Assuntos
Antioxidantes , Carpas , Animais , Antioxidantes/metabolismo , Lisina , Ácido Glutâmico , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Carpas/genética , Carpas/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Dieta/veterinária , Dipeptídeos/genética , Dipeptídeos/metabolismo , Expressão Gênica , Ração Animal/análise , Proteínas de Peixes/genéticaRESUMO
Non-alcoholic fatty liver disease (NAFLD) manifests as a persistent liver ailment marked by the excessive buildup of lipids within the hepatic organ accompanied by inflammatory responses and oxidative stress. Alanyl-glutamine (AG), a dipeptide comprising alanine and glutamine, is commonly employed as a nutritional supplement in clinical settings. This research aims to evaluate the impact of AG on NAFLD triggered by a high-fat diet (HFD), while concurrently delving into the potential mechanisms underlying its effects. The results presented herein demonstrate a notable reduction in the elevated body weight, liver mass, and liver index induced by a HFD upon AG administration. These alterations coincide with the amelioration of liver injury and the attenuation of hepatic histological advancement. Furthermore, AG treatment manifests a discernible diminution in oil-red-O-stained regions and triglyceride (TG) levels within the liver. Noteworthy alterations encompass lowered plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDLC) concentrations, coupled with elevated high-density lipoprotein cholesterol (HDLC) concentrations. The mitigation of hepatic lipid accumulation resultant from AG administration is aligned with the downregulation of ACC1, SCD1, PPAR-γ, and CD36 expression, in conjunction with the upregulation of FXR and SHP expression. Concomitantly, AG administration leads to a reduction in the accumulation of F4/80-positive macrophages within the liver, likely attributable to the downregulated expression of MCP-1. Furthermore, AG treatment yields a decline in hepatic MDA levels and a concurrent increase in the activities of SOD and GPX. A pivotal observation underscores the effect of AG in rectifying the imbalance of gut microbiota in HFD-fed mice. Consequently, this study sheds light on the protective attributes of AG against HFD-induced NAFLD through the modulation of gut microbiota composition.
Assuntos
Microbioma Gastrointestinal , Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Hepatopatia Gordurosa não Alcoólica/metabolismo , Dieta Hiperlipídica/efeitos adversos , Disbiose/metabolismo , Fígado/metabolismo , Dipeptídeos/farmacologia , Dipeptídeos/uso terapêutico , Dipeptídeos/metabolismo , Colesterol/metabolismo , Camundongos Endogâmicos C57BLRESUMO
High concentrations of ethanol could cause intracellular oxidative stress in yeast, which can lead to ethanol-oxidation cross-stress. Antioxidant dipeptides are effective in maintaining cell viability and stress tolerance under ethanol-oxidation cross-stress. In this study, we sought to elucidate how antioxidant dipeptides affect the yeast cell wall and membrane defense systems to enhance stress tolerance. Results showed that antioxidant dipeptide supplementation reduced cell leakage of nucleic acids and proteins by changing cell wall components under ethanol-oxidation cross-stress. Antioxidant dipeptides positively modulated the cell wall integrity pathway and up-regulated the expression of key genes. Antioxidant dipeptides also improved the cell membrane integrity by increasing the proportion of unsaturated fatty acids and regulating the expression of key fatty acid synthesis genes. Moreover, the addition of antioxidant dipeptides significantly (p < 0.05) increased the content of ergosterol. Ala-His (AH) supplementation caused the highest content of ergosterol, with an increase of 23.68 ± 0.01% compared to the control, followed by Phe-Cys (FC) and Thr-Tyr (TY). These results revealed that the improvement of the cell wall and membrane functions of antioxidant dipeptides was responsible for enhancing the ethanol-oxidation cross-stress tolerance of yeast.
Assuntos
Antioxidantes , Saccharomyces cerevisiae , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Parede Celular/metabolismo , Membrana Celular/metabolismo , Etanol/metabolismo , Ergosterol , Dipeptídeos/farmacologia , Dipeptídeos/metabolismoRESUMO
Proteinogenic dipeptides, with few known exceptions, are products of protein degradation. Dipeptide levels respond to the changes in the environment, often in a dipeptide-specific manner. What drives this specificity is currently unknown; what likely contributes is the activity of the different peptidases that cleave off the terminal dipeptide from the longer peptides. Dipeptidases that degrade dipeptides to amino acids, and the turnover rates of the "substrate" proteins/peptides. Plants can both uptake dipeptides from the soil, but dipeptides are also found in root exudates. Dipeptide transporters, members of the proton-coupled peptide transporters NTR1/PTR family, contribute to nitrogen reallocation between the sink and source tissues. Besides their role in nitrogen distribution, it becomes increasingly clear that dipeptides may also serve regulatory, dipeptide-specific functions. Dipeptides are found in protein complexes affecting the activity of their protein partners. Moreover, dipeptide supplementation leads to cellular phenotypes reflected in changes in plant growth and stress tolerance. Herein we will review the current understanding of dipeptides' metabolism, transport, and functions and discuss significant challenges and future directions for the comprehensive characterization of this fascinating but underrated group of small-molecule compounds.
Assuntos
Aminoácidos , Dipeptídeos , Dipeptídeos/química , Dipeptídeos/metabolismo , Transporte Biológico , Aminoácidos/metabolismo , NitrogênioRESUMO
L-Alanyl-L-Glutamine (Ala-Gln) is a common parenteral nutritional supplement. In our previous study, the recombinant whole-cell catalyst Escherichia coli BL21(DE3) overexpressing α-amino acid ester acyltransferase (BPA) to produce Ala-Gln has high activity and has been applied to large-scale production experiments. However, the degradation of Ala-Gln is detected under prolonged incubation, and endogenous broad-spectrum dipeptidase may be the primary cause. In this study, a CRISPR-Cas9 method was used to target pepA, pepB, pepD, pepN, dpp, and dtp to knock out one or more target genes. The deletion combination was optimized, and a triple knockout strain BL21(DE3)-ΔpepADN was constructed. The degradation performance of the knockout chassis was measured, and the results showed that the degradation rate of Ala-Gln was alleviated by 48% compared with the control. On this basis, BpADNPA (BPA-ΔpepADN) was built, and the production of Ala-Gln was 129% of the BPA's accumulation, proving that the ΔpepADN knockout conducive to the accumulation of dipeptide. This study will push forward the industrialization process of Ala-Gln production by whole-cell catalyst Escherichia coli expressing α-amino acid ester acyltransferase. KEY POINTS: ⢠Endogenous dipeptidase knockout alleviates the degradation of Ala-Gln by the chassis ⢠The balanced gene knockout combination is pepA, pepD, and pepN ⢠The accumulation of Ala-Gln with BpADNPA was 129% of the control.
Assuntos
Aminoácidos , Dipeptidases , Aminoácidos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Dipeptidases/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Técnicas de Inativação de Genes , Dipeptídeos/metabolismo , Glutamina/metabolismoRESUMO
The dipeptide carnosine is a physiologically important molecule in the human body, commonly found in skeletal muscle and brain tissue. Beta-alanine is a limiting precursor of carnosine and is among the most used sports supplements for improving athletic performance. However, carnosine, its metabolite N-acetylcarnosine, and the synthetic derivative zinc-L-carnosine have recently been gaining popularity as supplements in human medicine. These molecules have a wide range of effects-principally with anti-inflammatory, antioxidant, antiglycation, anticarbonylation, calcium-regulatory, immunomodulatory and chelating properties. This review discusses results from recent studies focusing on the impact of this supplementation in several areas of human medicine. We queried PubMed, Web of Science, the National Library of Medicine and the Cochrane Library, employing a search strategy using database-specific keywords. Evidence showed that the supplementation had a beneficial impact in the prevention of sarcopenia, the preservation of cognitive abilities and the improvement of neurodegenerative disorders. Furthermore, the improvement of diabetes mellitus parameters and symptoms of oral mucositis was seen, as well as the regression of esophagitis and taste disorders after chemotherapy, the protection of the gastrointestinal mucosa and the support of Helicobacter pylori eradication treatment. However, in the areas of senile cataracts, cardiovascular disease, schizophrenia and autistic disorders, the results are inconclusive.
Assuntos
Carnosina , Humanos , Carnosina/uso terapêutico , Antioxidantes/metabolismo , Suplementos Nutricionais , Dipeptídeos/metabolismo , Músculo Esquelético/metabolismo , beta-Alanina/farmacologia , beta-Alanina/metabolismoRESUMO
This work aimed to investigate the effect of aurantiamide (Aur) in promoting the M2 polarization of microglial cells to improve the cognitive ability of mice with Alzheimer's disease (AD). The M2 polarization of BV2 cells was induced by interleukin-4 (IL-4) treatment.Aur promoted the M2 polarization of BV2 cells, and up-regulated the expression of CD206 and SOCS3. In the meantime, it increased TGF-ß1, Arg-1 and IL-10 levels, and promoted the polarization of JAK1-STAT6. Treatment with STAT6 inhibitor antagonized the effect of Aur. Besides, the cognitive ability of AD mice was improved after Aur treatment, meanwhile, the expression of CD206 was up-regulated, while that of IBA-1 was down-regulated. Aur promotes the M2 polarization of microglial cells to improve the cognitive ability of AD mice, and such effect is related to the STAT6 signal.
Assuntos
Doença de Alzheimer , Microglia , Camundongos , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , CogniçãoRESUMO
High altitude is an environmental stress that is accompanied with numerous adverse biological responses, including skeletal muscle weakness and muscle protein loss. Skeletal muscle wasting is an important clinical problem, progressing to critical illness, associated with increased morbidity and mortality. The present study explores the protective efficacy of endogenous dipeptide, carnosine (CAR), supplementation in ameliorating skeletal muscle protein loss under hypobaric hypoxia (HH). Male Sprague-Dawley rats (n = 5) were randomly divided into control group, HH-exposed group (3 days HH exposure equivalent to 7,620 m), and HH-exposed rats supplemented with carnosine (3 days; 150 mg/kg b.w, orally) (HH + CAR). HH-exposed rats supplemented with CAR ameliorated HH-induced oxidative protein damage, lipid peroxidation, and maintained pro-inflammatory cytokines levels. HH-associated muscle protein degradative pathways, including calpain, ubiquitination, endoplasmic reticulum stress, autophagy, and apoptosis were also regulated in carnosine-supplemented rats. Further, the muscle damage marker, the levels of serum creatine phosphokinase were also reduced in HH + CAR co-supplemented rats which proved the protective efficacy of CAR against hypobaric hypoxia-induced muscle protein loss. Altogether, CAR supplementation ameliorated HH-induced skeletal muscle protein loss via performing multifaceted ways, mainly by maintaining redox homeostasis and proteostasis in skeletal muscle.
Assuntos
Carnosina , Proteostase , Animais , Carnosina/metabolismo , Carnosina/farmacologia , Suplementos Nutricionais , Dipeptídeos/metabolismo , Estresse do Retículo Endoplasmático , Hipóxia/tratamento farmacológico , Hipóxia/metabolismo , Masculino , Músculo Esquelético/metabolismo , Estresse Oxidativo/fisiologia , Ratos , Ratos Sprague-DawleyRESUMO
Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation (MC). Fungal conidiation can shift between the two patterns, which involves a large number of genes in the regulation of this process. In this study, we investigated the role of a dipeptidase gene pepdA in conidiation pattern shift in Metarhizium acridum, which is upregulated in MC pattern compared to typical conidiation. Results showed that disruption of the pepdA resulted in a shift of conidiation pattern from MC to typical conidiation. Metabolomic analyses of amino acids showed that the levels of 19 amino acids significantly changed in ΔpepdA mutant. The defect of MC in ΔpepdA can be rescued when nonpolar amino acids, α-alanine, ß-alanine, or proline, were added into sucrose yeast extract agar (SYA) medium. Digital gene expression profiling analysis revealed that PEPDA mediated transcription of sets of genes which were involved in hyphal growth and development, sporulation, cell division, and amino acid metabolism. Our results demonstrated that PEPDA played important roles in the regulation of MC by manipulating the levels of amino acids in M. acridum. IMPORTANCE Conidia, as the asexual propagules in many fungi, are the start and end of the fungal life cycle. In entomopathogenic fungi, conidia are the infective form essential for their pathogenicity. Filamentous fungi conduct two types of conidiation, typical conidiation from mycelia and microcycle conidiation. The mechanisms of the shift between the two conidiation patterns remain to be elucidated. In this study, we demonstrated that the dipeptidase PEPDA, a key enzyme from the insect-pathogenic fungus Metarhizium acridum for the hydrolysis of dipeptides, is associated with a shift of conidiation pattern. The conidiation pattern of the ΔpepdA mutant was restored when supplemented with the nonpolar amino acids rather than polar amino acids. Therefore, this report highlights that the dipeptidase PEPDA regulates MC by manipulating the levels of amino acids in M. acridum.
Assuntos
Dipeptidases/genética , Proteínas Fúngicas/genética , Metarhizium , Esporos Fúngicos/crescimento & desenvolvimento , Aminoácidos/genética , Dipeptidases/metabolismo , Dipeptídeos/metabolismo , Proteínas Fúngicas/metabolismo , Metarhizium/enzimologia , Metarhizium/genética , Metarhizium/fisiologiaRESUMO
A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is linked to the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Unconventional translation of the hexanucleotide repeat expansion generates five dipeptide repeat proteins (DPRs). The molecular mechanism underlying the DPR-linked neurotoxicity is under investigation. In this study, using cell-based models, we show that poly-proline-arginine DPR (poly-PR), the most neurotoxic DPR in vitro, binds to adenosine deaminase acting on RNA (ADAR)1p110 and ADAR2 and inhibits their RNA editing activity. We further show that poly-PR impairs cellular stress response that is mediated by ADAR1p110. These results together suggest that the poly-PR-mediated inhibition of the ADAR activity contributes to C9-ALS/FTD-linked neurotoxicity.
Assuntos
Adenosina Desaminase/genética , Arginina/genética , Proteína C9orf72/genética , Prolina/genética , Proteínas de Ligação a RNA/genética , Adenosina Desaminase/metabolismo , Animais , Arginina/metabolismo , Proteína C9orf72/metabolismo , Dipeptídeos/genética , Dipeptídeos/metabolismo , Células HeLa , Humanos , Camundongos , Neurônios/metabolismo , Prolina/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
Inflammatory bowel disease (IBD) is a chronic intestinal disorder threatening human health. Di-peptide alanyl-glutamine (Ala-Gln) has various beneficial effects on gut health. However, its role and functional mechanism in treating IBD are still not clear. Therefore, the protective effects of Ala-Gln and glutamine (Gln) on dextran sulfate sodium- (DSS-) induced colitic mice were investigated in this study. The results showed that oral supplementation of Ala-Gln or Gln significantly attenuated the colitis symptoms in mice, including body weight loss, colon length, disease activity index, histological scores, and tissue apoptosis. The concentrations of interleukin- (IL-) 1ß, IL-6, tumor necrosis factor-α, and myeloperoxidase were significantly decreased, while the concentrations of immunoglobulins (IgA, IgG, and IgM) and superoxide dismutase were significantly increased by Ala-Gln or Gln supplementation. The expression of occludin and peptide transporter 1 (PepT1) was significantly increased by Ala-Gln or Gln. Interestingly, Ala-Gln had better beneficial effects than Gln in alleviating colitis. In addition, 16S rDNA sequencing showed that the DSS-induced shifts of the microbiome (community diversity, evenness, richness, and composition) in the mouse colon were restored by Gln and Ala-Gln, including Lactobacillus, Bacteroides_acidifaciens, Bacteroidales, Firmicutes, Clostridia, Helicobacter, and Bacteroides. Correspondingly, the functions of the microflora metabolism pathways were also rescued by Ala-Gln, including fatty acid metabolism, membrane transporters, infectious diseases, and immune system. In conclusion, the results revealed that Ala-Gln can prevent colitis through PepT1, enhancing the intestinal barrier and modulating gut microbiota and microflora metabolites.
Assuntos
Colite/etiologia , Dipeptídeos/metabolismo , Microbioma Gastrointestinal/imunologia , Sulfatos/efeitos adversos , Animais , Colite/fisiopatologia , Humanos , Doenças Inflamatórias Intestinais , Masculino , CamundongosRESUMO
Soybean meal (SBM) inclusion in aquaculture diets has been found to negatively affect growth and induce intestinal inflammation in fish. The objective of this study was to determine the effect of health-promoting dipeptide supplementation into SBM-based feeds on growth performance, intestinal health, and muscle free amino acid composition, an indicator of dietary amino acid availability, in a zebrafish model. There were five treatment groups in this study. The first group ((+) Control) received a fishmeal-based diet. The second group ((-) Control) received SBM-based diet. The last three groups (Ala-Glu, Car, and Ans) were fed SBM-based diets, supplemented with alanyl-glutamine, carnosine, and anserine respectively. The Ala-Glu and Car groups experienced a significantly higher weight gain than the (-) Control group, weighing 35.38% and 33.96% more, respectively at the conclusion of the study. There were no significant differences in gene expression among the groups, but Ala-Glu had the highest expression of both nutrient absorption genes measured, PepT1 and fabp2. Ala-Glu had significantly longer intestinal villi, and a significantly higher villus length-to-width ratio than the (-) Control group. The Car group had a significantly higher post-prandial tissue concentration of lysine, compared to the (-) Control group. The increase in villus surface area and expression of nutrient absorption genes represent an improvement in intestinal absorptive capacity in the Ala-Glu group. The results from this study provide support for the use of alanyl-glutamine and carnosine supplementation as a means of improving growth performance of zebrafish fed with a high level SBM-based diet.
Assuntos
Ração Animal/análise , Dieta , Glycine max/química , Intestinos/efeitos dos fármacos , Peixe-Zebra/fisiologia , Animais , Aquicultura , Carnosina/farmacologia , Suplementos Nutricionais , Dipeptídeos/metabolismo , Dipeptídeos/farmacologia , Mucosa Intestinal/metabolismo , Proteínas de Plantas/metabolismoRESUMO
PURPOSE: Chronic ß-alanine supplementation leads to increased levels of muscle histidine-containing dipeptides. However, the majority of ingested ß-alanine is, most likely, degraded by two transaminases: GABA-T and AGXT2. In contrast to GABA-T, the in vivo role of AGXT2 with respect to ß-alanine metabolism is unknown. The purpose of the present work is to investigate if AGXT2 is functionally involved in ß-alanine homeostasis. METHODS: Muscle histidine-containing dipeptides levels were determined in AGXT2 overexpressing or knock-out mice and in human subjects with different rs37369 genotypes which is known to affect AGXT2 activity. Further, plasma ß-alanine kinetic was measured and urine was obtained from subjects with different rs37369 genotypes following ingestion of 1400 mg ß-alanine. RESULT: Overexpression of AGXT2 decreased circulating and muscle histidine-containing dipeptides (> 70% decrease; p < 0.05), while AGXT2 KO did not result in altered histidine-containing dipeptides levels. In both models, ß-alanine remained unaffected in the circulation and in muscle (p > 0.05). In humans, the results support the evidence that decreased AGXT2 activity is not associated with altered histidine-containing dipeptides levels (p > 0.05). Additionally, following an acute dose of ß-alanine, no differences in pharmacokinetic response were measured between subjects with different rs37369 genotypes (p > 0.05). Interestingly, urinary ß-alanine excretion was 103% higher in subjects associated with lower AGXT2 activity, compared to subjects associated with normal AGXT2 activity (p < 0.05). CONCLUSION: The data suggest that in vivo, ß-alanine is a substrate of AGXT2; however, its importance in the metabolism of ß-alanine and histidine-containing dipeptides seems small.
Assuntos
Carnosina/metabolismo , Transaminases/metabolismo , beta-Alanina/metabolismo , Adulto , Animais , Carnosina/genética , Dipeptídeos/genética , Dipeptídeos/metabolismo , Genótipo , Histidina/genética , Histidina/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculos/metabolismo , Transaminases/genética , Adulto Jovem , beta-Alanina/genéticaRESUMO
Purification of DPP-IV enzyme from porcine serum, is presented in this study for the first time. The high molecular weight DPP-IV from porcine serum was fractioned using Sephadex G-75 gel filtration followed by DEAE Sephadex anion exchange and Sephadex G-100 gel filtration chromatography columns with a final yield of 11.25%. The SDS-PAGE of the purified sample showed a single band of molecular mass nearing 160 kDa. Distinct single band was observed after PAS staining confirmed it to be a glycoprotein. The purified enzyme showed an optimum pH and temperature of 8 and 37 °C, respectively. The enzyme effectively cleaved fluorogenic substrate Gly-Pro-AMC with Km and Vmax of 4.578 µM and 90.84 nmoles/min, respectively. Purified DPP-IV activity was inhibited by Diprotin A with an IC50 value of 8.473 µM. Among the three plant extracts used to study DPP-IV inhibition, the aqueous hot extract of Terminalia chebula showed the highest inhibition of 87.19%, followed by the aqueous cold extract of Momordica carantia, ( 31.6%) and Azadirachta indica (34.16%) at the concentration of 25 µg.
Assuntos
Dipeptídeos/metabolismo , Dipeptidil Peptidase 4/isolamento & purificação , Ensaios Enzimáticos/métodos , Oligopeptídeos/farmacologia , Animais , Dipeptidil Peptidase 4/sangue , Dipeptidil Peptidase 4/química , Inibidores da Dipeptidil Peptidase IV/farmacologia , Cinética , Peso Molecular , Especificidade por Substrato , SuínosRESUMO
Although, existing methods have been successful in predicting phage (or bacteriophage) virion proteins (PVPs) using various types of protein features and complex classifiers, such as support vector machine and naïve Bayes, these two methods do not allow interpretability. However, the characterization and analysis of PVPs might be of great significance to understanding the molecular mechanisms of bacteriophage genetics and the development of antibacterial drugs. Hence, we herein proposed a novel method (PVPred-SCM) based on the scoring card method (SCM) in conjunction with dipeptide composition to identify and characterize PVPs. In PVPred-SCM, the propensity scores of 400 dipeptides were calculated using the statistical discrimination approach. Rigorous independent validation test showed that PVPred-SCM utilizing only dipeptide composition yielded an accuracy of 77.56%, indicating that PVPred-SCM performed well relative to the state-of-the-art method utilizing a number of protein features. Furthermore, the propensity scores of dipeptides were used to provide insights into the biochemical and biophysical properties of PVPs. Upon comparison, it was found that PVPred-SCM was superior to the existing methods considering its simplicity, interpretability, and implementation. Finally, in an effort to facilitate high-throughput prediction of PVPs, we provided a user-friendly web-server for identifying the likelihood of whether or not these sequences are PVPs. It is anticipated that PVPred-SCM will become a useful tool or at least a complementary existing method for predicting and analyzing PVPs.
Assuntos
Bacteriófagos/metabolismo , Biologia Computacional/métodos , Proteínas Virais/química , Vírion/metabolismo , Aminoácidos/metabolismo , Bases de Dados de Proteínas , Dipeptídeos/metabolismo , Internet , Pontuação de Propensão , Reprodutibilidade dos TestesRESUMO
PURPOSE: The aim of this study was to evaluate the efficacy and safety of Lu-PSMA-617 radioligand therapy in metastatic castration-resistant prostate cancer (mCRPC). METHODS: In this prospective, single-arm, single-institutional study, 90 mCRPC patients with progressive disease (PD) on second-line hormonal therapy and/or docetaxel chemotherapy were recruited for the study. All patients underwent diagnostic Ga-PSMA-HBED-CC PET/CT, prior to inclusion for therapy. Included patients underwent Lu-PSMA-617 therapy at 8- to 12-weekly intervals. The primary end point was to assess the overall survival. The secondary and cosecondary end points included biochemical response assessment as per the Prostate Cancer Working Group 3 criteria, progression-free survival, radiological and molecular response criteria, clinical response, safety profile, and disease control rates. All the outcome parameters were evaluated in 90 patients except for the radiographic and molecular response, which was evaluated in 69 patients. RESULTS: The median age of patients was 66.5 years (range, 30-88 years). The median activity administered per cycle was 3.7 to 8 GBq ranging from 1 to 7 cycles, and patients were followed up over a median duration of 28 months. At 2- to 3-month interval after the first therapy and the end of the assessment, greater than 50% decline in prostate-specific antigen was observed in 32.2% and 45.5%, respectively. Univariate analysis did not reveal any variables such as prior therapies, laboratory parameters, concomitant hormonal therapy, and SUV patient parameters associated with prostate-specific antigen decline. Radiographic response by diagnostic CT revealed partial remission in 23% (16/69), stable disease in 54% (37/69), and PD in 23% (16/69) of patients. Molecular tumor response by PET Response Criteria in Solid Tumor 1 criteria revealed 19 (27.5%) of 69 patients with partial remission, 30 (43.5%) of 69 with stable disease, and 20 (29%) of 69 with PD. The disease control rates according to the radiographic and molecular response were 77% and 71%, respectively. The median overall survival and median progression-free survivals were 14 and 11.8 months, respectively. Toxicities related to radioligand therapy were low and transient with no serious adverse effects. CONCLUSIONS: Lu-PSMA-617 radionuclide therapy is a safe and effective approach to the treatment of mCRPC patients.
Assuntos
Dipeptídeos/efeitos adversos , Dipeptídeos/uso terapêutico , Compostos Heterocíclicos com 1 Anel/efeitos adversos , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Neoplasias de Próstata Resistentes à Castração/radioterapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Dipeptídeos/metabolismo , Ácido Edético/análogos & derivados , Compostos Heterocíclicos com 1 Anel/metabolismo , Humanos , Ligantes , Lutécio , Masculino , Pessoa de Meia-Idade , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Estudos Prospectivos , Antígeno Prostático Específico , Neoplasias de Próstata Resistentes à Castração/diagnóstico por imagem , Neoplasias de Próstata Resistentes à Castração/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Resultado do TratamentoRESUMO
Bivalent chemical degraders provide a catalytic route to selectively degrade disease-associated proteins. By linking target-specific ligands with E3 ubiquitin ligase recruiting ligands, these compounds facilitate targeted protein ubiquitination and degradation by the proteasome. Due to the complexity of this multistep mechanism, the development of effective degrader molecules remains a difficult, lengthy, and unpredictable process. Since degraders are large heterobifunctional molecules, the efficacy of these compounds may be limited by poor cell permeability, and an efficient and reliable method to quantify the cell permeability of these compounds is lacking. Herein, we demonstrate that by the addition of a chloroalkane tag on the BRD4 specific degrader, MZ1, cell permeability can be quantified via the chloroalkane penetration assay. By extending this analysis to individual components of the degrader molecule, we have obtained structure-permeability relationships that will be informative for future degrader development, particularly as degraders move into the clinic as potential therapeutics.
Assuntos
Dipeptídeos/química , Dipeptídeos/metabolismo , Compostos Heterocíclicos com 3 Anéis/química , Compostos Heterocíclicos com 3 Anéis/metabolismo , Hidrocarbonetos/química , Proteólise/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/metabolismo , Técnicas Biossensoriais , Proteínas de Ciclo Celular/química , Linhagem Celular , Permeabilidade da Membrana Celular , Avaliação Pré-Clínica de Medicamentos , Humanos , Ligantes , Estrutura Molecular , Relação Estrutura-Atividade , Fatores de Transcrição/química , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND: Triple-negative breast cancer has extremely high risk of relapse due to the lack of targeted therapies, intra- and inter-tumoral heterogeneity, and the inherent and acquired resistance to therapies. In this study, we evaluate the potential of prostate-specific membrane antigen (PSMA) as target for radio-ligand therapy (RLT). METHODS: Tube formation was investigated after incubation of endothelial HUVEC cells in tumor-conditioned media and monitored after staining using microscopy. A binding study with 68Ga-labeled PSMA-addressing ligand was used to indicate targeting potential of PSMA on tumor-conditioned HUVEC cells. For mimicking of the therapeutic application, tube formation potential and vitality of tumor-conditioned HUVEC cells were assessed following an incubation with radiolabeled PSMA-addressing ligand [177Lu]-PSMA-617. For in vivo experiments, NUDE mice were xenografted with triple-negative breast cancer cells MDA-MB231 or estrogen receptor expressing breast cancer cells MCF-7. Biodistribution and binding behavior of [68Ga]-PSMA-11 was investigated in both tumor models at 30 min post injection using µPET. PSMA- and CD31-specific staining was conducted to visualize PSMA expression and neovascularization in tumor tissue ex vivo. RESULTS: The triple-negative breast cancer cells MDA-MB231 showed a high pro-angiogenetic potential on tube formation of endothelial HUVEC cells. The induced endothelial expression of PSMA was efficiently addressed by radiolabeled PSMA-specific ligands. 177Lu-labeled PSMA-617 strongly impaired the vitality and angiogenic potential of HUVEC cells. In vivo, as visualized by µPET, radiolabeled PSMA-ligand accumulated specifically in the triple-negative breast cancer xenograft MDA-MB231 (T/B ratio of 43.3 ± 0.9), while no [68Ga]-PSMA-11 was detected in the estrogen-sensitive MCF-7 xenograft (T/B ratio of 1.1 ± 0.1). An ex vivo immunofluorescence analysis confirmed the localization of PSMA on MDA-MB231 xenograft-associated endothelial cells and also on TNBC cells. CONCLUSIONS: Here we demonstrate PSMA as promising target for two-compartment endogenous radio-ligand therapy of triple-negative breast cancer.
Assuntos
Radioisótopos de Gálio/uso terapêutico , Glutamato Carboxipeptidase II/antagonistas & inibidores , Lutécio/uso terapêutico , Radioisótopos/uso terapêutico , Neoplasias de Mama Triplo Negativas/radioterapia , Animais , Antígenos de Superfície/metabolismo , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/fisiologia , Vasos Sanguíneos/efeitos da radiação , Linhagem Celular Tumoral , Meios de Cultivo Condicionados/farmacologia , Dipeptídeos/metabolismo , Dipeptídeos/uso terapêutico , Ácido Edético/análogos & derivados , Ácido Edético/metabolismo , Ácido Edético/uso terapêutico , Isótopos de Gálio , Glutamato Carboxipeptidase II/metabolismo , Compostos Heterocíclicos com 1 Anel/metabolismo , Compostos Heterocíclicos com 1 Anel/uso terapêutico , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Ligantes , Células MCF-7 , Camundongos Nus , Oligopeptídeos/metabolismo , Oligopeptídeos/uso terapêutico , Antígeno Prostático Específico , Compostos Radiofarmacêuticos/uso terapêutico , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Matriptase and hepsin belong to the family of type II transmembrane serine proteases (TTSPs). Increased activity of these and the plasma protease, hepatocyte growth factor activator (HGFA), is associated with unregulated cell signaling and tumor progression through increased MET and RON kinase signaling pathways. These proteases are highly expressed in multiple solid tumors and hematological malignancies. Herein, we detail the synthesis and structure-activity relationships (SAR) of a dipeptide library bearing Arg α-ketobenozothiazole (kbt) warheads as novel inhibitors of HGFA, matriptase, and hepsin. We elucidated the substrate specificity for HGFA using positional scanning of substrate combinatorial libraries (PS-SCL), which was used to discover selective inhibitors of matriptase and hepsin. Using these selective inhibitors, we have clarified the specific role of hepsin in maintaining epithelial cell membrane integrity, known to be lost in breast cancer progression. These selective compounds are useful as chemical biology tools and for future drug discovery efforts.