RESUMEN
l-Carnosine (l-Car), an endogenous dipeptide presents in muscle and brain tissues of various vertebrates, has a wide range of application values. The enzymatic preparation of l-Car is a promising synthetic method because it avoids the protection and deprotection steps. In the present study, a dipeptidase gene (CpPepD) from Clostridium perfringens with high l-Car synthetic activity was cloned and characterized. In an effort to improve the performance of this enzyme, we carried out site saturation mutagenesis using CpPepD as the template. By the o-phthalaldehyde (OPA)-derived high throughput screening method, mutant A171S was obtained with 2.2-fold enhanced synthetic activity. The enzymatic properties of CpPepD and mutant A171S were investigated. Under the optimized conditions, 63.94â¯mM (14.46â¯gâ¯L-1) or 67.02â¯mM (15.16â¯gâ¯L-1) l-Car was produced at the substrate concentrations of 6â¯M ß-Ala and 0.2â¯M l-His using wild-type or mutant A171S enzyme, respectively. Although the mutation enhanced the enzyme activity, the reaction equilibrium was barely affected.
Asunto(s)
Carnosina , Clostridium perfringens , Dipeptidasas , Clostridium perfringens/enzimología , Clostridium perfringens/genética , Carnosina/metabolismo , Carnosina/química , Carnosina/análogos & derivados , Dipeptidasas/genética , Dipeptidasas/metabolismo , Dipeptidasas/química , Ingeniería de Proteínas/métodos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Mutagénesis Sitio-DirigidaRESUMEN
Carnosine is a natural bioactive dipeptide with important physiological functions widely used in food and medicine. Dipeptidase (PepD) from Serratia marcescens can catalyze the reverse hydrolytic reaction of ß-alanine with l-histidine to synthesize carnosine in the presence of Mn2+. However, it remains challenging to practice carnosine biosynthesis due to the low activity and high cost of the enzyme. Therefore, the development of biocatalysts with high activity and stability is of significance for carnosine synthesis. Here, we proposed to chelate Mn2+ to polyethylenimine (PEI) that induced rapid formation of calcium phosphate nanocrystals (CaP), and Mn-PEI@CaP was used for PepD immobilization via electrostatic interaction. Mn-PEI@CaP as the carrier enhanced the stability of the immobilized enzyme. Moreover, Mn2+ loaded in the carrier acted as an in situ activator of the immobilized PepD for facilitating the biocatalytic process of carnosine synthesis. The as-prepared immobilized enzyme (PepD-Mn-PEI@CaP) kept similar activity with free PepD plus Mn2+ (activity recovery, 102.5%), while exhibiting elevated thermal stability and pH tolerance. Moreover, it exhibited about two times faster carnosine synthesis than the free PepD system. PepD-Mn-PEI@CaP retained 86.8% of the original activity after eight cycles of batch catalysis without the addition of free Mn2+ ions during multiple cycles. This work provides a new strategy for the co-immobilization of PepD and Mn2+, which greatly improves the operability of the biocatalysis and demonstrates the potential of the immobilized PepD system for efficient carnosine synthesis.
Asunto(s)
Fosfatos de Calcio , Carnosina , Dipeptidasas , Enzimas Inmovilizadas , Manganeso , Nanopartículas , Polietileneimina , Carnosina/química , Carnosina/metabolismo , Polietileneimina/química , Manganeso/química , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Fosfatos de Calcio/química , Nanopartículas/química , Dipeptidasas/metabolismo , Dipeptidasas/química , Serratia marcescens/enzimología , BiocatálisisRESUMEN
Vaccines represent a pivotal health advancement for preventing infection. However, because carrier systems with repeated administration can invoke carrier-targeted immune responses that diminish subsequent immune responses (e.g., PEG antibodies), there is a continual need to develop novel vaccine platforms. Zinc carnosine microparticles (ZnCar MPs), which are composed of a one-dimensional coordination polymer formed between carnosine and the metal ion zinc, have exhibited efficacy in inducing an immune response against influenza. However, ZnCar MPs' limited suspendability hinders clinical application. In this study, we address this issue by mixing mannan, a polysaccharide derived from yeast, with ZnCar MPs. We show that the addition of mannan increases the suspendability of this promising vaccine formulation. Additionally, since mannan is an adjuvant, we illustrate that the addition of mannan increases the antibody response and T cell response when mixed with ZnCar MPs. Mice vaccinated with mannan + OVA/ZnCar MPs had elevated serum IgG and IgG1 levels in comparison to vaccination without mannan. Moreover, in the mannan + OVA/ZnCar MPs vaccinated group, mucosal washes demonstrated increased IgG, IgG1, and IgG2c titers, and antigen recall assays showed enhanced IFN-γ production in response to MHC-I and MHC-II immunodominant peptide restimulation, compared to the vaccination without mannan. These findings suggest that the use of mannan mixed with ZnCar MPs holds potential for subunit vaccination and its improved suspendability further promotes clinical translation.
Asunto(s)
Carnosina , Mananos , Vacunas de Subunidad , Zinc , Mananos/química , Mananos/administración & dosificación , Mananos/inmunología , Animales , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Zinc/química , Zinc/administración & dosificación , Carnosina/administración & dosificación , Carnosina/química , Femenino , Inmunoglobulina G/sangre , Ratones , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Ovalbúmina/inmunología , Ovalbúmina/administración & dosificación , Ratones Endogámicos C57BL , Polímeros/química , Polímeros/administración & dosificación , Ratones Endogámicos BALB C , Portadores de Fármacos/químicaRESUMEN
Treatments for organ-confined prostate cancer include external beam radiation therapy, radical prostatectomy, radiotherapy/brachytherapy, cryoablation and high-intensity focused ultrasound. None of these are cancer-specific and are commonly accompanied by side effects, including urinary incontinence and erectile dysfunction. Moreover, subsequent surgical treatments following biochemical recurrence after these interventions are either limited or affected by the scarring present in the surrounding tissue. Carnosine (ß-alanyl-L-histidine) is a histidine-containing naturally occurring dipeptide which has been shown to have an anti-tumorigenic role without any detrimental effect on healthy cells; however, its effect on prostate cancer cells has never been investigated. In this study, we investigated the effect of carnosine on cell proliferation and metabolism in both a primary cultured androgen-resistant human prostate cancer cell line, PC346Flu1 and murine TRAMP-C1 cells. Our results show that carnosine has a significant dose-dependent inhibitory effect in vitro on the proliferation of both human (PC346Flu1) and murine (TRAMP-C1) prostate cancer cells, which was confirmed in 3D-models of the same cells. Carnosine was also shown to decrease adenosine triphosphate content and reactive species which might have been caused in part by the increase in SIRT3 also shown after carnosine treatment. These encouraging results support the need for further human in vivo work to determine the potential use of carnosine, either alone or, most likely, as an adjunct therapy to surgical or other conventional treatments.
Asunto(s)
Braquiterapia , Carnosina , Disfunción Eréctil , Neoplasias de la Próstata , Masculino , Humanos , Animales , Ratones , Carnosina/farmacología , Carnosina/química , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/cirugía , Dipéptidos , Braquiterapia/efectos adversos , Disfunción Eréctil/etiologíaRESUMEN
Carnosine, an endogenous dipeptide, has been found to have a plethora of medicinal properties, such as antioxidant, antiageing, and chelating effects, but with one downside: a short half-life. Carnosinases and two hydrolytic enzymes, which remain enigmatic, are responsible for these features. Hence, here we emphasize why research is valuable for better understanding crucial concepts like ageing, neurodegradation, and cancerogenesis, given that inhibition of carnosinases might significantly prolong carnosine bioavailability and allow its further use in medicine. Herein, we explore the literature regarding carnosinases and present a short in silico analysis aimed at elucidating the possible recognition pattern between CN1 and its ligands.
Asunto(s)
Carnosina , Dipeptidasas , Humanos , Carnosina/química , Carnosina/metabolismo , Antioxidantes , Dipeptidasas/química , Dipeptidasas/metabolismo , EnvejecimientoRESUMEN
The lipid profile of skin is fundamental in the maintenance of the protective barrier against the external environment. Signaling and constitutive lipids of this large organ are involved in inflammation, metabolism, aging, and wound healing, such as phospholipids, triglycerides, FFA, and sphingomyelin. Skin exposure to ultraviolet (UV) radiation results in a photoaging process that is an accelerated form of aging. UV-A radiation deeply penetrates the dermis and promotes damage to DNA, lipids, and proteins by increasing the generation of reactive oxygen species (ROS). Carnosine, an endogenous ß-alanyl-L-histidine dipeptide, demonstrated antioxidant properties that prevent photoaging and modification of skin protein profiling, making carnosine a compelling ingredient to consider for use in dermatology. The aim of this research was to investigate the modification of skin lipidome after UV-A treatment in presence or not of topic administration of carnosine. Quantitative analyses based on high-resolution mass spectrometry of nude mice skin-extracted lipids resulted in several modifications of barrier composition after UV-A radiation, with or without carnosine treatment. In total, 328 out of 683 molecules showed significant alteration-262 after UV-A radiation and 126 after UV-A and carnosine treatment versus controls. Importantly, the increased oxidized TGs after UV-A radiation, responsible of dermis photoaging, were completely reverted by carnosine application to prevent the UV-A damage. Network analyses also showed that the production of ROS and the calcium and TNF signaling were modulated by UV-A and carnosine. In conclusion, lipidome analyses attested the carnosine activity to prevent the UV-A damage, reducing the lipid oxidation, the inflammation, and the dysregulation of lipid skin barrier.
Asunto(s)
Carnosina , Envejecimiento de la Piel , Enfermedades de la Piel , Animales , Ratones , Carnosina/farmacología , Carnosina/química , Ratones Desnudos , Especies Reactivas de Oxígeno/metabolismo , Lipidómica , Rayos Ultravioleta/efectos adversos , Fosfolípidos , InflamaciónRESUMEN
Carnosine and anserine were reported to inhibit tyrosine nitration. However, there are no reports on the nitration inhibitory activities of balenine, 2-oxo-carnosine, 2-oxo-anserine, and 2-oxo-balenine. We demonstrated for the first time that these compounds exhibit inhibitory activities against peroxynitrite-dependent tyrosine nitration. 2-Oxo-imidazole dipeptides (2-oxo-IDPs) showed higher inhibitory activity than their precursor IDPs, thereby suggesting that 2-oxo-IDPs may be effective against nitrative stress-related diseases.
Asunto(s)
Carnosina , Carnosina/farmacología , Carnosina/química , Anserina , Ácido Peroxinitroso , Dipéptidos/farmacología , Dipéptidos/química , Imidazoles/farmacología , Imidazoles/química , TirosinaRESUMEN
It is generally accepted that carnosine (ß-alanyl-L-histidine) content is higher in glycolytic than in oxidative muscle fibres, but the underlying mechanisms responsible for this difference remain to be elucidated. A first study to better understand potential mechanisms involved was undertaken (1) to determine whether differences in the expression of carnosine-related enzymes (CARNS1, CNDP2) and transporters (SLC6A6, SLC15A3, SLC15A4, SLC36A1) exist between oxidative and glycolytic myofibres and (2) to study the effect of carnosine on myoblast proliferative growth and on carnosine-related gene expression in cultured myoblasts isolated from glycolytic and oxidative muscles. Immunohistochemistry analyses were conducted to determine the cellular localization of carnosine-related proteins. Laser-capture microdissection and qPCR analyses were performed to measure the expression of carnosine-related genes in different myofibres isolated from the longissimus dorsi muscle of ten crossbred pigs. Myogenic cells originating from glycolytic and oxidative muscles were cultured to assess the effect of carnosine (0, 10, 25 and 50 mM) on their proliferative growth and on carnosine-related gene expression. The mRNA abundance of CNDP2 and of the studied carnosine transporters was higher in oxidative than in glycolytic myofibres. Since carnosine synthase (CARNS1) mRNA abundance was not affected by either the fibre type or the addition of carnosine to myoblasts, its transcriptional regulation would not be the main process by which carnosine content differences are determined in oxidative and glycolytic muscles. The addition of carnosine to myoblasts leading to a dose-dependent increase in SLC15A3 transcripts, however, suggests a role for this transporter in carnosine uptake and/or efflux to maintain cellular homeostasis.
Asunto(s)
Carnosina , Porcinos , Animales , Carnosina/análisis , Carnosina/química , Carnosina/metabolismo , Músculo Esquelético/metabolismo , Fibras Musculares Esqueléticas/metabolismo , ARN Mensajero/genéticaRESUMEN
This study quantified the nutritional components and imidazole dipeptide levels of commercially available meats (beef, pork, and duck), and their effects on taste were quantified via taste recognition devices. Although meat and its products are considered high-risk diets, meat components, such as imidazole dipeptides, exert bioregulatory functions. Further, considering their bioregulatory function, commercial meats' antioxidant activity and vascular endothelial function were examined. Characteristic variations in nutritional components were observed depending on the type and part of meat analyzed. These components affected the taste and texture of meat. The main imidazole dipeptides detected were anserine (duck meat) and carnosine (beef and pork). Meat with larger quantities of total imidazole dipeptide demonstrated better sensory test results. Therefore, anserine and carnosine effects on taste were determined using a taste recognition device; carnosine alone produced a noticeably bitter taste, whereas adding anserine reduced bitterness and enhanced umami taste. In a few cases, cooking enhanced the quantity of carnosine and/or anserine and their antioxidant activities. We demonstrated the ability of imidazole dipeptides, particularly anserine, to improve nitric oxide production in vascular endothelial cells. This study provides essential information for health-conscious consumers to develop high-quality, functional meat products.
Asunto(s)
Carnosina , Carne de Cerdo , Carne Roja , Animales , Bovinos , Porcinos , Dipéptidos , Carnosina/química , Anserina , Patos , Gusto , Células Endoteliales , Carne/análisis , Antioxidantes , Imidazoles/farmacologíaRESUMEN
Human carnosinases (CNs) are dimeric dipeptidases in the metallopeptidase M20 family. Two isoforms of carnosinases (Zn2+ -containing carnosinase 1 (CN1) found in serum and Mn2+ -carnosinase 2 (CN2) in tissue) were identified. Both CNs cleave histidine-containing (Xaa-His) dipeptides such as carnosine where CN2 was found to accept a broader spectrum of substrates. A loss of CN function, resulting in a high carnosine concentration, reduces risk for diabetes and neurological disorders. Although several studies on CN activities and its Michaelis complex were conducted, all shed the light on CN1 activity where the CN2 data is limited. Also, the molecular details on CN1 and CN2 similarity and dissimilarity in structure and function remain unclear. Thus, in this work, molecular dynamics (MD) simulations were employed to study structure and dynamics of human CN1 and CN2 in comparison. The results show that the different catalytic ability of both CNs is due to their pocket size and environment. CN2 can accept a wider range of substrate due to the wider mouth of a binding pocket. The L1 loop seems to play a role in gating activity. Comparing to CN2, CN1 provides more electronegative entrance, more wettability, and higher stability of catalytic metal ion-pair in the active site which allow more efficient water-mediated catalysis. The microscopic understanding obtained here can serve as a basis for CN inhibition strategies resulting in higher carnosine levels and consequently mitigating complications associated with diseases such as diabetes and neurological disorder.
Asunto(s)
Carnosina , Dipeptidasas , Humanos , Carnosina/química , Carnosina/metabolismo , Dipeptidasas/genética , Dipéptidos/química , Simulación de Dinámica MolecularRESUMEN
Imidazole dipeptides, histidine-containing dipeptides, including carnosine (ß-alanyl-l-histidine), anserine (ß-alanyl-3-methyl-l-histidine), and balenine (ß-alanyl-1-methyl-l-histidine) in animal muscles have physiological functions, such as significant antioxidant and antifatigue effects. They are obtained by extraction from natural raw materials, including chicken and fish meat. However, using natural raw materials entails stable supply and mass production limitations. l-amino acid α-ligase (Lal) catalyzes the formation of various dipeptides from unprotected l-amino acids by conjugating with adenosine 5'-triphosphate (ATP) hydrolysis reaction. In this study, site-directed mutagenesis of Lal was applied to establish an efficient method for producing imidazole dipeptides by the enzymatic process. We significantly improved the conversion rate from substrate amino acids compared with wild-type Lal.
Asunto(s)
Aminoácidos , Carnosina , Animales , Aminoácidos/metabolismo , Ligasas/metabolismo , Histidina/genética , Dipéptidos/metabolismo , Carnosina/química , Anserina/metabolismo , Mutagénesis Sitio-Dirigida , ImidazolesRESUMEN
Skin hyperpigmentation is an aesthetic problem that leads to psychosocial issues. Thus, skin whitening agents from agro- and poultry-industrial co-products are considered high economic value ingredients of interest for sustainable application. Therefore, this study aimed to determine the cosmeceutical potential of anserine/carnosine-rich chicken extract (ACCE) from the Thai native chicken Pradu Hang Dam Mor Kor 55 (PD) meat. The chemical composition was identified and quantified using the HPLC-UV method. Then, the antioxidation potential of the extract was compared to that of L-anserine and L-carnosine, using 1,1-diphenyl-2-picrylhydrazyl assay and shikonin-induced production of reactive oxygen species in CCD-986Sk cell models, and the anti-melanogenesis effect in the MNT-1 melanoma cell line model was investigated. Furthermore, related mechanisms were identified using colorimetric tyrosinase assay and the Western blot technique. The ACCE was composed of L-anserine and L-carnosine as two major constituents. In a dose-dependent manner, ACCE, L-anserine, and L-carnosine manifested significant antioxidation potential and significant reduction of melanin production. Activation of the extracellular signal-regulated kinase (ERK) signaling pathway and inhibition of tyrosinase activity of ACCE were demonstrated as the mechanisms of the anti-melanogenesis effect. In conclusion, ACCE has been revealed as a potential cosmeceutical agent due to its antioxidation and anti-melanogenic activity in association with L-anserine and L-carnosine composition and biomolecular regulating ability. Therefore, further studies and development should be considered to support the utilization of anserine/carnosine-rich chicken extract in the cosmetic industry for economic value creation and sustainability.
Asunto(s)
Carnosina , Cosmecéuticos , Animales , Anserina/química , Carnosina/química , Pollos/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Monofenol Monooxigenasa/metabolismo , Tailandia , Antioxidantes/farmacología , Antioxidantes/metabolismo , Transducción de SeñalRESUMEN
As a biologically active peptide, L-carnosine has been widely used in the pharmaceutical, cosmetic and health care industries due to its various physiological properties. However, relatively little research is available regarding L-carnosine's enzymatic synthesis function. In this study, a potential enzyme sequence with the function of carnosine synthesizing was screened out using the ancestral sequence reconstruction (ASR) technique. Identified with L-carnosine synthesis activity, this enzyme was further confirmed using autoproteolytic phenomenon via Western blot and N-terminal sequencing. After purification, the enzymatic properties of LUCA-DmpA were characterized. The melting temperature (Tm) and denaturation enthalpy (ΔH) of LUCA-DmpA were 60.27 ± 1.24 °C and 1306.00 ± 26.73 kJ·mol-1, respectively. Circular dichroism (CD) spectroscopy results showed that this ancestral enzyme was composed of α-helix (35.23 ± 0.06%), ß-sheet (11.06 ± 0.06%), ß-turn (23.67 ± 0.06%) and random coil (32.03 ± 0.06%). The enzyme was characterized with the optimal temperature and pH of 45 °C and 9.0, respectively. Notably, LUCA-DmpA was also characterized with remarkable pH tolerance based on the observation of more than 85% remaining enzymatic activity after incubation at different pH buffers (pH = 6-11) for 12 h. Additionally, rather than being improved or inhibited by metal ions, its enzymatic activity was found to be promoted by introducing organic solvent with a larger log P value. Based on these homology modeling results, the screened LUCA-DmpA is suggested to have further optimization potential, and thereafter to be offered as a promising candidate for real industrial applications.
Asunto(s)
Carnosina , Aminopeptidasas , Carnosina/química , Iones , Preparaciones Farmacéuticas , SolventesRESUMEN
Carcinine is a natural imidazole-containing peptide derivative. It is widely used in the cosmetics industry as anti-aging supplement with antioxidant, anti-glycation and glycation reversal functions, and it also has a notable pharmacological effect as anti-tumor drug and in protection against retinopathy. However, a technological method for synthesis and production of carcinine has not been established. In this study, a whole-cell transformation system converting ß-alanine and histamine to carcinine by the enzymes Ebony and phosphopantetheine transferase (Sfp) has been developed. The results revealed that the catalytic efficiency of the strain containing the fusion protein of Ebony and Sfp (Sfp-glycine-serine-glycine-Ebony, SGE) in Escherichia coli W3110 (WSGE strain) is significantly higher (7.45 mM) than the combinatorial strain of pET28a-ebony and pACYCDuet-sfp in E. coli BL21(DE3) (BSE strain) (2.17 mM). Under the optimal reaction conditions (25 â, pH 7.0, 12.5 g/L wet cells, 20 mM ß-alanine and 40 mM histamine), the carcinine can be quickly synthesized within 24 h up to a concentration of 22.63 mM. To achieve a continuous and efficient conversion of the precursors, a batch-feeding catalysis was designed. With this system, ß-alanine (40 mM) and histamine (40 mM) could be completely transformed to carcinine (40.34 mM) in 36 h with a productivity of 0.204 g/L h reaching a titer of 7.34 g/L. Hence, the batch-feeding whole-cell biocatalysis is a promising technology for the high yield production of carcinine which can promote the industrial production of carcinine.
Asunto(s)
Carnosina , Escherichia coli , Histamina , Biotransformación , Carnosina/análogos & derivados , Carnosina/química , Escherichia coli/genética , Escherichia coli/metabolismo , Glicina/metabolismo , Histamina/metabolismo , Ingeniería Metabólica/métodos , beta-Alanina/metabolismoRESUMEN
L-Carnosine is a natural biologically active dipeptide with critical physiological functions, such as antioxidant, antiglycation, and cytoplasmic buffering properties. Direct enzymatic synthesis is a promising way for L-carnosine production. In this study, a new aminopeptidase (gene_236976) with synthetic activity toward L-carnosine was identified by a metagenome mining approach from deep-sea sediment and functionally expressed in Escherichia coli. The enzyme shared a low identity of 14.3% with reported L-carnosine dipeptidase (SmPepD) from Serratia marcescens. ß-Alanine methyl ester was proven to be the best substrate for the synthesis, and no ATP was needed for the enzymatic reaction. The enzyme activity was increased by structure-guided rational design. Only the mutant of G310 site gave positive results, and G310A mutant showed the best performance among the site-direct saturation mutagenesis, indicating that the additional CH3 group of mutant G310A was the main factor affecting the enzymatic activity. The engineered enzyme produced about 10 mM L-carnosine was produced from substrates of 50 mM ß-alanine methyl ester and 50 mM L-histidine, under a tentatively optimized condition. This study enriched the enzyme resources for developing the microbial synthesis process of L-carnosine production.
Asunto(s)
Carnosina , Antioxidantes , Carnosina/química , Carnosina/fisiología , Dipéptidos , Histidina , MetagenomaRESUMEN
Carnosine and anserine are abundant peptides found in the skeletal muscle and nervous system in many vertebrates. Several in vitro and in vivo studies have demonstrate that exogenously administered carnosine improves exercise performance. Furthermore, carnosine is an antioxidant and antifatigue supplement. However, the physiological functions of endogenous carnosine and its related histidine-containing dipeptides in a living organism remain unclear. We aimed to clarify the physiological roles of endogenous carnosine by investigating the characteristics of carnosine synthase gene-deficient mice and the effects of carnosine on skeletal muscle protein metabolism. We discovered that carnosine and anserine were undetectable in the skeletal muscle of carnosine synthase knockout mice. We also quantified protein gene expression and enzyme levels in muscle protein metabolism. Gene and protein levels of the muscle protein synthesizer insulin-like growth factor-1 (IGF-1) and the degrading enzyme cathepsin B were markedly lower in carnosine synthase gene-deficient mice than those in wild-type mice. The amount of 3-methylhistidine (a marker for muscle proteolysis) in forced exercise and the weight of the gastrocnemius muscle were considerably lower in carnosine synthase gene-deficient mice than in wild-type mice. Consequently, we showed that carnosine deficiency affects weight maintenance and protein metabolism in skeletal muscle, suggesting that carnosine regulates skeletal muscle protein metabolism.
Asunto(s)
Anserina , Carnosina , Péptido Sintasas/metabolismo , Animales , Carnosina/química , Dipéptidos/metabolismo , Ratones , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismoRESUMEN
The naturally occurring dipeptide carnosine (ß-alanyl-l-histidine) has beneficial effects in different diseases. It is also frequently used as a food supplement to improve exercise performance and because of its anti-aging effects. Nevertheless, after oral ingestion, the dipeptide is not detectable in human serum because of rapid degradation by serum carnosinase. At the same time, intact carnosine is excreted in urine up to five hours after intake. Therefore, an unknown compartment protecting the dipeptide from degradation has long been hypothesized. Considering that erythrocytes may constitute this compartment, we investigated the uptake and intracellular amounts of carnosine in human erythrocytes cultivated in the presence of the dipeptide and human serum using liquid chromatography-mass spectrometry. In addition, we studied carnosine's effect on ATP production in red blood cells and on their response to oxidative stress. Our experiments revealed uptake of carnosine into erythrocytes and protection from carnosinase degradation. In addition, no negative effect on ATP production or defense against oxidative stress was observed. In conclusion, our results for the first time demonstrate that erythrocytes can take up carnosine, and, most importantly, thereby prevent its degradation by human serum carnosinase.
Asunto(s)
Adenosina Trifosfato/metabolismo , Carnosina/metabolismo , Dipeptidasas/metabolismo , Eritrocitos/metabolismo , Estrés Oxidativo , Suero/enzimología , Carnosina/química , Eritrocitos/patología , HumanosRESUMEN
It has been found that the self-assembly of nonfluorescent peptides can generate fluorescent peptide nanoparticles (f-PNPs) to perform multiple functions, including drug delivery and imaging and tracking therapeutic agents. Both pharmacologically inactive peptides and tumor-targeting peptides have been explored to construct biocompatible f-PNPs; however, the application of this technology in delivering antitumor peptides has never been reported. Herein, the self-assembly of an antitumor dipeptide, carnosine, into fluorescent carnosine nanoparticles (f-Car NPs) in the presence of zinc ions is demonstrated. The generated f-Car NPs exhibit fluorescence in the visible and near-infrared (NIR) ranges for fluorescence tracing in vitro and in vivo. On the other hand, the f-Car NPs minimize the contact between the dipeptide and the serum, which overcomes the dipeptide instability resulted from inefficient antitumor activity. In addition, the preparation of f-Car NPs does not introduce extra carrier materials, so the f-Car NPs exhibit biocompatibility to normal fibroblast cells in vitro and negligible toxicity against major organs in vivo. This study provides a new peptide drug delivery strategy with NIR fluorescence tracing ability.
Asunto(s)
Antineoplásicos/uso terapéutico , Carnosina/uso terapéutico , Colorantes Fluorescentes/uso terapéutico , Nanopartículas del Metal/uso terapéutico , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológico , Animales , Antineoplásicos/química , Antineoplásicos/efectos de la radiación , Antineoplásicos/toxicidad , Carnosina/química , Carnosina/efectos de la radiación , Carnosina/toxicidad , Línea Celular Tumoral , Femenino , Colorantes Fluorescentes/química , Colorantes Fluorescentes/efectos de la radiación , Colorantes Fluorescentes/toxicidad , Fluorometría/métodos , Humanos , Rayos Infrarrojos , Nanopartículas del Metal/química , Nanopartículas del Metal/efectos de la radiación , Nanopartículas del Metal/toxicidad , Ratones Endogámicos BALB C , Nanomedicina Teranóstica/métodos , Zinc/químicaRESUMEN
Colored compounds formed by the Maillard reaction of carnosine with xylose or glucose were investigated in this study. Yellow pigments showing an absorption maximum at 450 nm were found in a heated solution of carnosine with xylose at pH 5.0. These pigments were then isolated and identified as dicarnosyl-dipyrrolones A and B. The generation of dipyrrolones in the absence of lysine suggests that dipyrrolone pigments can be formed by pentose as well as every amino compound such as amino acids, peptides and proteins possessing a free amino group. Analysis of α-dicarbonyls using LC-MS/MS showed that pentosone, 1-deoxypentosone, 3-deoxypentosone (3-DP), and methylglyoxal were predominantly generated via degradation of Amadori compounds. Also, a potential formation pathway of dypyrrolones was established, indicating that an Amadori compound that could form 3-DP is likely to play a role as a main precursor for dipyrrolones.
Asunto(s)
Ácidos/química , Carnosina/química , Reacción de Maillard , Pentosas/química , Pigmentos Biológicos/química , Pirroles/químicaRESUMEN
The ability of histidine to scavenge sugar-derived 1,2-dicarbonyl compounds was investigated using aqueous methanolic model systems containing histidine or histamine in the presence of glucose, methylglyoxal, or glyoxal. The samples were prepared either at room temperature (RT) or at 150 °C and analyzed using ESI-qTOF-MS/MS and isotope labeling technique. Replacing glucose with [U-13C6]glucose allowed the identification of glucose carbon atoms incorporated in the products. Various sugar-generated carbonyl compounds ranging in size from C1 to C6 were captured by histidine or histamine. The majority of the fragments incorporated were either C3 or C2 units originating from glyoxal (C2) or methylglyoxal (C3). The ESI-qTOF-MS/MS analysis indicated that histamine could react with either of the two carbonyl carbons of methylglyoxal utilizing the α-amino group and/or the imidazolium moiety. Furthermore, when histidine was added to 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP) generating model system, it completely suppressed the formation of PhIP due to scavenging of phenylacetaldehyde.