RESUMO
PADI4 is one of the human isoforms of a group of enzymes intervening in the conversion of arginine to citrulline. It is involved in the development of several types of tumors, as well as other immunological illnesses, such as psoriasis, multiple sclerosis, or rheumatoid arthritis. PADI4 auto-citrullinates in several regions of its sequence, namely in correspondence of residues Arg205, Arg212, Arg218, and Arg383. We wanted to study whether the citrullinated moiety affects the conformation of nearby regions and its binding to intact PADI4. We designed two series of synthetic peptides comprising either the wild-type or the relative citrullinated versions of such regions - i.e., a first series of peptides comprising the first three arginines, and a second series comprising Arg383. We studied their conformational properties in isolation by using fluorescence, far-ultraviolet (UV) circular dichroism (CD), and 2D1H NMR. Furthermore, we characterized the binding of the wild-type and citrullinated peptides in the two series to the intact PADI4, by using isothermal titration calorimetry (ITC), fluorescence, and biolayer interferometry (BLI), as well as by molecular docking simulations. We observed that citrullination did not alter the local conformational propensities of the isolated peptides. Nevertheless, for all the peptides in the two series, citrullination slowed down the kinetic koff rates of the binding reaction to PADI4, probably due to differences in electrostatic effects compared to the presence of arginine. The affinities of PADI4 for unmodified peptides were slightly larger than those of the corresponding citrullinated ones in the two series, but they were all within the same range, indicating that there were no relevant variations in the thermodynamics of binding due to sequence effects. These results highlight details of the self-citrullination of PADI4 and, more generally, of possible auto-catalytic mechanisms taking place in vivo for other citrullinating enzymes or, alternatively, in proteins undergoing citrullination passively.
Assuntos
Citrulinação , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas , Proteína-Arginina Desiminase do Tipo 4/metabolismo , Proteína-Arginina Desiminase do Tipo 4/química , Humanos , Desiminases de Arginina em Proteínas/metabolismo , Desiminases de Arginina em Proteínas/química , Conformação Proteica , Peptídeos/química , Peptídeos/metabolismo , Citrulina/química , Citrulina/metabolismo , Ligação Proteica , Sequência de AminoácidosRESUMO
Peptide identification by positive electrospray ionization (ES+) tandem mass spectrometry (MS/MS) is a well-established strategy in proteomics. Several research groups reported the usefulness of negative electrospray ionization (ES-) for gaining complementary structural information on peptides and their post-translational modifications (PTM) compared to ES+. Fragmentation of citrullinated peptides has not been previously explored in ES-. In this study, 9 peptides containing citrulline residues were investigated in ES- by stepwise collision energy-dependent measurements on a QTOF instrument and a Q-Orbitrap instrument. Our results of high resolution and mass accuracy show the favored citrulline-selective loss of HNCO from these peptide precursors and their fragmentsâsimilarly to that in ES+âalong with y-NH3/z, c, c-NH3/b sequence ions. Loss of HNCO from citrullinated peptides in ES- and a proposed mechanism for the reaction have been described here for the first time. HNCO loss intensities from precursors were generally even higher than that in ES+. Interestingly, the most intense fragments corresponded to neutral losses from sequence ions while intact sequence ions were usually minor components of the spectra. High-intensity ions related to cleavages N-terminal to Asp and Glu residues that have been previously reported were also observed. On the other hand, a relatively high number of peaks were observed, possibly due to internal fragmentation and/or scrambling events. While (ES-) MS/MS spectra always require manual inspection and the annotation may be ambiguous, the favorable loss of HNCO and the preferable cleavage N-terminal to Asp residues can be used to differentiate between citrullinated/deamidated sequences.
Assuntos
Citrulina , Espectrometria de Massas em Tandem , Espectrometria de Massas em Tandem/métodos , Citrulina/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Peptídeos/química , Ânions , ÍonsRESUMO
Post-translational modification of arginine to citrulline is catalyzed by members of the peptidylarginine deiminase (PAD) family. Dysregulation of this catalysis is a significant driver of the pathogenesis of numerous inflammatory diseases, including cancer. However, dysregulation of PAD activity has not been examined in breast cancer with respect to hormone receptor status. In this study, we measured PAD enzyme levels using Western blotting and investigated protein citrullination using a mass spectrometry-based proteomics approach in primary estrogen receptor negative (ER-) or positive (ER+) breast tumor and matched adjacent normal tissue. Our findings reveal 72 and 41 citrullinated proteins in ER- tumor and adjacent healthy tissue, respectively, where 20 of these proteins are common between the two groups. We detected 64 and 49 citrullinated proteins in ER+ tumor and adjacent healthy tissue, respectively, where 32 proteins are common. Interestingly, upon comparison of ER- and ER+ tumor tissue, only 32 citrullinated proteins are shared between the two and the rest are unique to the tumor's receptor status. Using the STRING database for protein-protein interaction network analysis, these proteins are involved in protein-folding events (i.e., heat shock proteins) in ER- samples and blood-clotting events (i.e., fibulin) in ER+ samples. Constituents of the extracellular matrix structure (i.e., collagen and fibrinogen) were found in both. Herein, we establish evidence that supports the role of this unique post-translational modification in breast cancer biology. Finally, to aid drug discovery against citrullination, we developed a liquid chromatography-ultraviolet method to measure PAD enzymatic activity and optimized glucagon-like peptide II to quantitatively measure the ability of PADs to citrullinate its substrate.
Assuntos
Neoplasias da Mama , Citrulinação , Humanos , Feminino , Proteínas/metabolismo , Desiminases de Arginina em Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Citrulina/química , Hidrolases/químicaRESUMO
In recent years, arginine deiminase (ADI, EC 3.5.3.6) has attracted much attention as a biocatalyst that produces the functional amino acid l-citrulline from l-arginine and also as an anticancer enzyme. Here, we identified and characterized a putative ADI from the thermophilic bacterium Halothermothrix orenii. The H. orenii ADI (H-ADI) protein was expressed in Escherichia coli BL21(DE3) with a specific activity of 91.8 U/mg protein at 55°C and pH 6.5. The enzyme remained at 74% relative activity after incubation at 45°C for 180 min, only 25% at 50°C. The melting temperature was 56°C. H-ADI is not a metal-requiring enzyme; Ni2+ slightly improved the catalytic activity. The Km and Vmax for l-arginine were 55.5 mM and 156.8 µmol/min/mg protein, respectively. Moreover, three residues (Arg183, Arg237, and His273) were key to the formation of l-citrulline, as analyzed by alanine-scanning mutagenesis. Finally, the enzymatic synthesis of l-citrulline was carried out at 50°C with a conversion ratio reaching 99.03%. Together, these findings show that H-ADI is a promising biocatalyst for the production of l-citrulline.
Assuntos
Citrulina , Hidrolases , Citrulina/química , Citrulina/metabolismo , Hidrolases/química , ArgininaRESUMO
This work describes the enhancement of a novel antitumor therapeutic platform that combines advantages from small-molecule drug conjugates (SMDCs) and antibody drug conjugates (ADCs). Valine-citrulline (VCit) dipeptide linkers are commonly used cathepsin B cleavable linkers for ADCs. However, the instability of these linkers in mouse serum makes translating efficacy data from mouse to human more challenging. Replacing the VCit linker with glutamic acid-valine-citrulline (EVCit) has been reported to enhance the stability of ADCs in mouse serum. However, the effect of EVCit linker on the stability of SMDCs has not been reported. Here, we report that incorporating the EVCit linker in prostate-specific membrane antigen-targeting SMDCs, equipped with the transthyretin ligand AG10, resulted in conjugates with lower toxicity, an extended half-life, and superior therapeutic efficacy to docetaxel in a xenograft mouse model of prostate cancer. This should make SMDCs' preclinical toxicity and efficacy data from mice more reliable for predicting human results.
Assuntos
Antineoplásicos , Imunoconjugados , Animais , Humanos , Camundongos , Anticorpos Monoclonais/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Linhagem Celular Tumoral , Citrulina/química , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Imunoconjugados/química , Ligantes , Pré-Albumina , ValinaRESUMO
The use of tandem mass spectrometry (MS/MS) is a fundamental prerequisite of reliable protein identification and quantification in mass-spectrometry-based proteomics. In bottom-up and middle-down proteomics, proteins are identified by the characteristic fragments of their constituting peptides. Post-translational modifications (PTMs) often further complicate proteome analyses. Citrullination is an increasingly studied PTM converting arginines to citrullines (Cit, X) and is implicated in several autoimmune and neurological diseases as well as different types of cancer. Confirmation of citrullination is known to be very challenging since it results in the same molecular mass change as Asn/Gln deamidation. In this study, we explore which MS/MS characteristics can be used for the reliable identification of citrullination. We synthesized several peptides incorporating Cit residues that model enzymatic cleavages of different proteins with verified or putative citrullination. Collision-induced dissociation was used to investigate the energy dependence of Byonic and Mascot scores and confirmed sequence coverage (CSC) along with the neutral loss of HNCO characteristic to citrulline side chains. We found that although the recommended values (19-45 V) for ramped collision energy settings cover the optimal Mascot, Byonic, or %CSC scores effectively, the diagnostic HNCO loss from precursors and fragments may reach their maximum intensities at lower and higher collision energies, respectively. Therefore, we suggest broadening the ramp range to â¼5-60 V to obtain more favorable identification rates for citrullinated peptides. We also found that Byonic was more successful in correctly identifying citrullinated peptides with deamidated residues than Mascot.
Assuntos
Peptídeos , Espectrometria de Massas em Tandem , Citrulina/química , Peptídeos/química , Processamento de Proteína Pós-Traducional , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
BACKGROUND: Microglia are the primary phagocytes of the central nervous system and are responsible for removing damaged myelin following demyelination. Previous investigations exploring the consequences of myelin phagocytosis on microglial activation overlooked the biochemical modifications present on myelin debris. Such modifications, including citrullination, are increased within the inflammatory environment of multiple sclerosis lesions. METHODS: Mouse cortical myelin isolated by ultracentrifugation was citrullinated ex vivo by incubation with the calcium-dependent peptidyl arginine deiminase PAD2. Demyelination was induced by 6 weeks of cuprizone (0.3%) treatment and spontaneous repair was initiated by reversion to normal chow. Citrullinated or unmodified myelin was injected into the primary motor cortex above the cingulum bundle at the time of reversion to normal chow and the consequent impact on remyelination was assessed by measuring the surface area of myelin basic protein-positive fibers in the cortex 3 weeks later. Microglial responses to myelin were characterized by measuring cytokine release, assessing flow cytometric markers of microglial activation, and RNAseq profiling of transcriptional changes. RESULTS: Citrullinated myelin induced a unique microglial response marked by increased tumor necrosis factor α (TNFα) production both in vitro and in vivo. This response was not induced by unmodified myelin. Injection of citrullinated myelin but not unmodified myelin into the cortex of cuprizone-demyelinated mice significantly inhibited spontaneous remyelination. Antibody-mediated neutralization of TNFα blocked this effect and restored remyelination to normal levels. CONCLUSIONS: These findings highlight the role of post-translation modifications such as citrullination in the determination of microglial activation in response to myelin during demyelination. The inhibition of endogenous repair induced by citrullinated myelin and the reversal of this effect by neutralization of TNFα may have implications for therapeutic approaches to patients with inflammatory demyelinating disorders.
Assuntos
Quelantes , Citrulina/química , Cuprizona , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/patologia , Microglia/metabolismo , Bainha de Mielina/química , Fator de Necrose Tumoral alfa/biossíntese , Animais , Células Cultivadas , Citocinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microinjeções , Córtex Motor , Proteína Básica da MielinaRESUMO
Citrullination is an enzyme-catalyzed post-translational modification (PTM) that is essential for a host of biological processes, including gene regulation, programmed cell death, and organ development. While this PTM is required for normal cellular functions, aberrant citrullination is a hallmark of autoimmune disorders as well as cancer. Although aberrant citrullination is linked to human pathology, the exact role of citrullination in disease remains poorly characterized, in part because of the challenges associated with identifying the specific arginine residues that are citrullinated. Tandem mass spectrometry is the most precise method for uncovering sites of citrullination; however, due to the small mass shift (+0.984 Da) that results from citrullination, current database search algorithms commonly misannotate spectra, leading to a high number of false-positive assignments. To address this challenge, we developed an automated workflow to rigorously and rapidly mine proteomic data to unambiguously identify the sites of citrullination from complex peptide mixtures. The crux of this streamlined workflow is the ionFinder software program, which classifies citrullination sites with high confidence on the basis of the presence of diagnostic fragment ions. These diagnostic ions include the neutral loss of isocyanic acid, which is a dissociative event that is unique to citrulline residues. Using the ionFinder program, we have mapped the sites of autocitrullination on purified protein arginine deiminases (PAD1-4) and mapped the global citrullinome in a PAD2-overexpressing cell line. The ionFinder algorithm is a highly versatile, user-friendly, and open-source program that is agnostic to the type of instrument and mode of fragmentation that are used.
Assuntos
Citrulinação/fisiologia , Mineração de Dados/métodos , Proteômica/métodos , Algoritmos , Arginina/metabolismo , Citrulinação/genética , Citrulina/química , Citrulina/genética , Citrulina/metabolismo , Análise de Dados , Gerenciamento de Dados/métodos , Humanos , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
Rheumatoid arthritis (RA) is a systemic inflammatory autoimmune disease which affects primarily the joints. Peptides of several proteins have shown an effect in some experimental animal models of RA. We investigated arthritis development in male DBA/1 mice which were injected with bovine collagen II (bCII) and human fibrinogen (hFib) on days 0 and 21, leading to stable and reproducible disease induction in 100% of immunized mice (FIA-CIA). In a second study, two bCII-derived peptides were given three times in the course of 6 weeks after FIA-CIA induction to test for impact on arthritis. Mice were scored weekly for arthritis and anti-citrullinated peptide antibodies (ACPAs) were determined in the sera taken on days 0, 14, 35, 56 and 84. Histology of the hind paws was performed at the end of the experiment. Intravenous administration of peptide 90578, a novel fructosylated peptide derived from the immunodominant T cell epitope of bCII, at a dosage of 1 mg/kg resulted in significant beneficial effects on clinical outcome parameters and on the arthritis histology scores which was sustained over 12 weeks. Survival tended to be improved in peptide 90578-treated mice. Intravenous administration of pure soluble peptide 90578 without adjuvants is a promising approach to treat RA, with treatment starting at a time when ACPAs are already present. The results complement existing data on peptide "vaccination" of healthy animals, or on treatment using recombinant peptide expressing virus or complex biological compounds.
Assuntos
Artrite Reumatoide/imunologia , Artrite/imunologia , Artrite/metabolismo , Epitopos de Linfócito T/química , Frutose/química , Peptídeos/química , Animais , Antígenos de Diferenciação de Linfócitos B , Autoimunidade , Bovinos , Citrulina/química , Colágeno Tipo II/química , Antígenos de Histocompatibilidade Classe II , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos DBA , Peptídeos CíclicosRESUMO
Peptidylarginine deiminases (PAD) enzymes were initially characterized in uteri, but since then little research has examined their function in this tissue. PADs post-translationally convert arginine residues in target proteins to citrulline and are highly expressed in ovine caruncle epithelia and ovine uterine luminal epithelial (OLE)-derived cell line. Progesterone (P4) not only maintains the uterine epithelia but also regulates the expression of endometrial genes that code for proteins that comprise the histotroph and are critical during early pregnancy. Given this, we tested whether P4 stimulates PAD-catalyzed histone citrullination to epigenetically regulate expression of the histotroph gene insulin-like growth factor binding protein 1 (IGFBP1) in OLE cells. 100 nM P4 significantly increases IGFBP1 mRNA expression; however, this increase is attenuated by pre-treating OLE cells with 100 nM progesterone receptor antagonist RU486 or 2 µM of a pan-PAD inhibitor. P4 treatment of OLE cells also stimulates citrullination of histone H3 arginine residues 2, 8, and 17 leading to enrichment of the ovine IGFBP1 gene promoter. Since PAD2 nuclear translocation and catalytic activity require calcium, we next investigated whether P4 triggers calcium influx in OLE cells. OLE cells were pre-treated with 10 nM nicardipine, an L-type calcium channel blocker, followed by stimulation with P4. Using fura2-AM imaging, we found that P4 initiates a rapid calcium influx through L-type calcium channels in OLE cells. Furthermore, this influx is necessary for PAD2 nuclear translocation and resulting citrullination of histone H3 arginine residues 2, 8, and 17. Our work suggests that P4 stimulates rapid calcium influx through L-type calcium channels initiating PAD-catalyzed histone citrullination and an increase in IGFBP1 expression.
Assuntos
Citrulinação , Citrulina/química , Regulação da Expressão Gênica/efeitos dos fármacos , Histonas/química , Progesterona/farmacologia , Útero/metabolismo , Animais , Endométrio/efeitos dos fármacos , Endométrio/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Proteína 1 de Ligação a Fator de Crescimento Semelhante à Insulina , Gravidez , Progestinas/farmacologia , Ovinos , Útero/efeitos dos fármacosRESUMO
BACKGROUND: Specific modifications to carriers to achieve targeted delivery of chemotherapeutics into malignant tissues are a critical point for efficient diagnosis and therapy. In this case, bovine serum albumin (BSA) was conjugated with cetuximab-valine-citrulline (vc)-doxorubicin (DOX) to target epidermal growth factor receptor (EGFR) and enable the release of drug in EGFR-overexpressed tumor cells. METHODS: Maleimidocaproyl-valine-citrulline-p-aminobenzylcarbonyl-p-nitrophenol (MC-Val-Cit-PAB-PNP) and DOX were used to synthesize MC-Val-Cit-PAB-DOX, which was further linked with cetuximab to prepare antibody-drug conjugates (ADCs). Then, the ADCs were adsorbed to the surface of the BSA nanoparticles (NPs), which were prepared by a desolvation method to obtain cetuximab-vc-DOX-BSA-NPs. The cetuximab-vc-DOX conjugates adsorbed on the surface of the BSA nanoparticles were determined and optimized by size exclusion chromatography. An in vitro cytotoxicity study was conducted using a colon carcinoma cell line with different EGFR-expression levels to test the selectivity of cetuximab-vc-DOX-NPs. RESULTS: The vc-DOX and cetuximab-vc-DOX conjugates were both synthesized successfully and their structural characteristics confirmed by 1H-NMR and SDS-PAGE. The MTT assay showed stronger cytotoxicity of cetuximab-vc-DOX-NPs versus control IgG-vc-DOX-NPs in EGFR-overexpressing RKO cells. Cellular binding and intracellular accumulation determined by flow cytometry and confocal laser scanning microscopy revealed the strong binding ability of cetuximab-vc-DOX-NPs to RKO cells. The in vivo imaging study demonstrated that cetuximab-vc-DOX-NPs exhibited higher fluorescent intensity in tumor tissues than non-decorated nanoparticles (IgG-vc-DOX-NPs). In vivo tumor inhibition and survival tests showed that cetuximab-vc-DOX-NPs revealed higher tumor inhibition efficacy and lower systemic toxicity than control IgG-vc-DOX- NPs. CONCLUSION: The obtained results emphasize that cetuximab-vc-DOX-NPs, with good tumor-targeting ability and low systemic toxicity, are a promising targeting system for drug delivery.
Assuntos
Cetuximab/uso terapêutico , Citrulina/química , Neoplasias Colorretais/tratamento farmacológico , Doxorrubicina/uso terapêutico , Receptores ErbB/metabolismo , Nanopartículas/química , Soroalbumina Bovina/química , Valina/química , Adsorção , Animais , Bovinos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Endocitose/efeitos dos fármacos , Humanos , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Camundongos , Tamanho da Partícula , Espectroscopia de Prótons por Ressonância Magnética , Distribuição Tecidual/efeitos dos fármacosRESUMO
Citrullination is a post-translational modification (PTM) that converts peptidyl-arginine into peptidyl-citrulline; citrullination is catalyzed by the protein arginine deiminases (PADs). This PTM is associated with several physiological processes, including the epigenetic regulation of gene expression, neutrophil extracellular trap formation, and DNA-damage induced apoptosis. Notably, aberrant protein citrullination is relevant to several autoimmune and neurodegenerative diseases and certain forms of cancer. As such, the PADs are promising therapeutic targets. In this review, we discuss recent advances in the development of PAD inhibitors and activity-based probes, the development and use of citrulline-specific probes in chemoproteomic applications, and methods to site-specifically incorporate citrulline into proteins.
Assuntos
Arginina/química , Citrulina/química , Desiminases de Arginina em Proteínas/metabolismo , Proteínas/química , Animais , Doenças Autoimunes/metabolismo , Catálise , Citrulinação , Epigênese Genética , Humanos , Neoplasias/metabolismo , Doenças Neurodegenerativas/metabolismo , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-TraducionalRESUMO
OBJECTIVE: To investigate the target delivery properties of RC48-ADC, a novel antibody drug conjugate (ADC) comprising cytotoxic monomethyl auristatin E (MMAE) and an anti-human epidermal growth factor receptor 2 (HER2) antibody tethered via valine-citrulline linker, in vitro and in vivo. MATERIALS AND METHODS: Dissociation rate of MMAE from RC48-ADC was used as an estimate of its stability in serum. Cytotoxicity of the antibody and RC48-ADC towards multiple cell lines was measured. Subcellular distribution of the drug was determined by fluorescence imaging. The mechanism of lysosome targeting was verified. Endocytic pathways of RC48-ADC were assessed by the cellular fluorescence intensity of fluorescently-labelled drugs. Intracellular and extracellular distribution of MMAE was analysed after RC48-ADC or MMAE administration to characterize MMAE release. The serum and tumour concentration of MMAE was compared after tail-vein injection of RC48-ADC into tumour-bearing mice. RESULTS: RC48-ADC was highly stable in human serum. HER2-overexpressed cell line SK-BR-3 proliferation was stronger when suppressed by RC48-ADC than by the naked antibody. Both RC48-ADC and naked antibody were internalized via caveolae-mediated and clathrin-mediated endocytosis and concentrated in lysosomes. Higher HER2 expression was associated with enhanced uptake and intracellular release of conjugated MMAE; free MMAE could kill tumour cells via the bystander effect. Although serum RC48-ADC concentration was higher than that in tumours, exposure of MMAE in tumours was ~200 times higher than in serum, which rationalized the reduced toxicity of RC48-ADC. CONCLUSIONS: In vitro and in vivo experiments confirmed the targeted transport and release of RC48-ADC; it could selectively deliver MMAE to the targeted HER2-positive cell or tumour tissue, which could reduce off-target toxicity and enhance anti-tumour potency in humans.
Assuntos
Anticorpos Monoclonais/farmacologia , Citrulina/farmacologia , Sistemas de Liberação de Medicamentos , Imunoconjugados/farmacologia , Oligopeptídeos/farmacologia , Valina/farmacologia , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citrulina/sangue , Citrulina/química , Feminino , Imunoconjugados/sangue , Imunoconjugados/química , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Oligopeptídeos/sangue , Oligopeptídeos/química , Receptor ErbB-2/genética , Células Tumorais Cultivadas , Valina/sangue , Valina/químicaRESUMO
Watermelon (Citrulus lantus) is an important horticultural crop which belongs to the Curcubitaceae family. The nutraceutical potential of watermelon has been illustrated by several researchers, which makes it a better choice of functional food. Watermelon has been used to treat various ailments, such as cardio-vascular diseases, aging related ailments, obesity, diabetes, ulcers, and various types of cancers. The medicinal properties of watermelon are attributed by the presence of important phytochemicals with pharmaceutical values such as lycopene, citrulline, and other polyphenolic compounds. Watermelon acts as vital source of l-citrulline, a neutral-alpha amino acid which is the precursor of l-arginine, an essential amino acid necessary for protein synthesis. Supplementation of l-citrulline and lycopene displayed numerous health benefits in in vitro and in vivo studies. Similarly, the dietary intake of watermelon has proven benefits as functional food in humans for weight management. Apart from the fruits, the extracts prepared from the seeds, sprouts, and leaves also evidenced medicinal properties. The present review provides a comprehensive overview of benefits of watermelon for the treatment of various ailments.
Assuntos
Citrullus/química , Suplementos Nutricionais , Compostos Fitoquímicos/química , Extratos Vegetais/farmacologia , Aminoácidos/química , Animais , Antioxidantes/química , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/prevenção & controle , Citrulina/química , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/prevenção & controle , Diabetes Mellitus/tratamento farmacológico , Diabetes Mellitus/prevenção & controle , Dieta , Frutas/química , Alimento Funcional , Horticultura , Humanos , Licopeno/química , Óxido Nítrico/metabolismo , Obesidade/tratamento farmacológico , Obesidade/prevenção & controle , Plantas Medicinais/metabolismoAssuntos
Aterosclerose/sangue , Diabetes Mellitus Tipo 2/sangue , Armadilhas Extracelulares , Ativação de Neutrófilo , Fumar/sangue , Idoso , Aterosclerose/epidemiologia , Citrulina/química , Comorbidade , Diabetes Mellitus Tipo 2/epidemiologia , Endotélio Vascular/patologia , Feminino , Histonas/química , Humanos , Molécula 1 de Adesão Intercelular/sangue , Masculino , Pessoa de Meia-Idade , Selectina-P/sangue , Placa Aterosclerótica/sangue , Placa Aterosclerótica/epidemiologia , Ruptura Espontânea , Fumar/epidemiologia , Molécula 1 de Adesão de Célula Vascular/sangueRESUMO
Dairy cow mastitis is a common bacterial infectious disease which seriously threatens the development of the dairy cow industry. Previous studies have found that increased IFN-γ expression in dairy cows makes dairy cows more susceptible to mastitis, but the underlying mechanism is still not known. In this study, we utilized the in vitro bovine mammary epithelial cells (BMECs) model to explore the molecular mechanism via transcriptome sequencing technology, immunofluorescence, and Western blotting. It was found that IFN-γ promoted the adhesion and invasion of Staphylococcus aureus to BMECs through increasing the expression of TLR4-mediated CCL5 in BMECs. IFN-γ increased the activity of arginase II and reduced the level of arginine in cells, while the addition of arginine inhibited the expression of TLR4 and CCL5. An invasion experiment in mice further validated that IFN-γ treatment significantly increased the bacterial load in mammary glands and blood. However, the colonization and diffusion of S. aureus were interestingly decreased after Arg supplement. These data reveal that increased IFN-γ reduces arginine levels and activates TLR4-CCL5 signaling, leading to enhanced susceptibility of BMECs to S. aureus. Our findings are helpful to understand the pathogenesis of dairy cow mastitis and provide a theoretical basis for improvement of mastitis resistance in dairy cows.
Assuntos
Arginina/metabolismo , Quimiocina CCL5/metabolismo , Células Epiteliais/metabolismo , Interferon gama/metabolismo , Glândulas Mamárias Animais/metabolismo , Staphylococcus aureus/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Arginina/química , Aderência Bacteriana , Bovinos , Citrulina/química , Feminino , Mastite/fisiopatologia , Camundongos , Ornitina/química , Ratos , Transdução de SinaisRESUMO
The GE81112 tetrapeptides are a small family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. With the exception of the 3-hydroxy-l-pipecolic acid unit, little is known about the biosynthetic origins of the non-proteinogenic amino acid monomers of the natural product family. Here, we elucidate the biogenesis of the 4-hydroxy-l-citrulline unit and establish the role of an iron- and α-ketoglutarate-dependent enzyme (Fe/αKG) in the pathway. Homology modelling and sequence alignment analysis further facilitate the rational engineering of this enzyme to become a specific 4-arginine hydroxylase. We subsequently demonstrate the utility of this engineered enzyme in the synthesis of a dipeptide fragment of the antibiotic enduracidin. This work highlights the value of applying a bioinformatics-guided approach in the discovery of novel enzymes and engineering of new catalytic activity into existing ones.
Assuntos
Citrulina/química , Hidroxilação/genética , Peptídeos/química , Pirrolidinas/síntese química , Biocatálise , Especificidade por SubstratoRESUMO
BACKGROUND: Hyperactivity of the mechanistic target of rapamycin complex 1 (mTORC1) is implicated in a variety of diseases such as cancer and diabetes. Treatment may benefit from effective mTORC1 inhibition, which can be achieved by preventing arginine from disrupting the cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1)-GTPase-activating proteins toward RAGS subcomplex 2 (GATOR2) complex through binding with CASTOR1. An attractive idea is to determine analogues of arginine that are as competent as arginine in binding with CASTOR1, but without disrupting the CASTOR1-GATOR2 interaction. MATERIALS AND METHODS: Molecular dynamics simulations were performed for binding of arginine analogues with CASTOR1 and binding free energy, hydrogen bond formation, and root mean squared deviation and root mean square fluctuation kinetics were then calculated. RESULTS: The binding free energy calculations revealed that Nα-acetyl-arginine, citrulline, and norarginine have sufficient binding affinity with CASTOR1 to compete with arginine. The hydrogen bond analysis revealed that norarginine, Nα-acetyl-arginine and D-arginine have proficient H-bonds that can facilitate their entering the narrow binding pocket. CONCLUSION: Norarginine and Nα-acetyl-arginine are the top drug candidates for mTORC1 inhibition, with Nα-acetyl-arginine being the best choice.
Assuntos
Arginina/análogos & derivados , Citrulina/química , Peptídeos e Proteínas de Sinalização Intracelular , Alvo Mecanístico do Complexo 1 de Rapamicina , Simulação de Dinâmica Molecular , Arginina/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/químicaRESUMO
Protein arginine deiminases (PADs) hydrolyze the side chain of arginine to form citrulline. Aberrant PAD activity is associated with rheumatoid arthritis, multiple sclerosis, lupus, and certain cancers. These pathologies established the PADs as therapeutic targets and multiple PAD inhibitors are known. Herein, we describe the first highly potent PAD1-selective inhibitors (1 and 19). Detailed structure-activity relationships indicate that their potency and selectivity is due to the formation of a halogen bond with PAD1. Importantly, these inhibitors inhibit histone H3 citrullination in HEK293TPAD1 cells and mouse zygotes with excellent potency. Based on this scaffold, we also developed a PAD1-selective activity-based probe that shows remarkable cellular efficacy and proteome selectivity. Based on their potency and selectivity we expect that 1 and 19 will be widely used chemical tools to understand PAD1 biology.
Assuntos
Citrulinação/efeitos dos fármacos , Citrulina/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteína-Arginina Desiminase do Tipo 1/antagonistas & inibidores , Animais , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/enzimologia , Células HEK293 , Histonas/química , Humanos , Isoenzimas , Camundongos , Proteína-Arginina Desiminase do Tipo 1/metabolismoRESUMO
Tandem mass spectrometry of peptides is of utmost importance in proteomics. Collision-induced dissociation usually generates y type fragment ion series from tryptic peptides, carrying information on their primary structure. Amino acid side chains or differences in their basicity could alter fragmentation processes considerably. The well-known proline effect is a cleavage preference at the N-terminus of proline residues in peptides, usually yielding a very abundant y ion while suppressing others. Previously, we reported a similar phenomenon occurring at the C-terminus of citrulline residues and coined the term Cit effect. To confirm the presence of Cit effect in large proteomic datasets, we analyzed 293 peptides containing Cit residues based on the human proteome database mining work of Lee et al. (2018). The occurrence of Cit effect was found to be 44%. Comparing bond scissions at the amide linkage between Cit-Zzz (citrulline followed by a specified residue) to Aaa1-Aaa2 (Aaa can be any residue except Cit), 5 Cit-Zzz cleavages were significantly (CL = 95.0%) more frequent in > 85% of the cases in terms of relative sequential base beak occurrence. We used Pro effect to compare with Cit effect and obtained very similar results. On the other hand, our study showed that Cit effect is slightly inferior in the overall incidence to Pro effect (50% vs. 33%, CL = 95%) among deiminated peptides when Pro residues were also present in the sequence. Our results suggest that Cit effect is a characteristic feature and a possible biasing factor of deiminated peptides which can confirm the position of citrullination sites.