RESUMEN
OBJECTIVES: To investigate genotype-phenotype characteristics and long-term prognosis of neonatal carbamoyl phosphate synthetase 1 (CPS1) deficiency among children through newborn screening in Zhejiang province. METHODS: The clinical and follow-up data of children with CPS1 deficiency detected through neonatal screening and confirmed by tandem mass spectrometry and genetic testing in Zhejiang Province Newborn Disease Screening Center from September 2013 to August 2023 were retrospectively analyzed. RESULTS: A total of 4 056 755 newborns were screened and 6 cases of CPS1 deficiency were diagnosed through phenotypic and genetic testing. Ten different variations of CPS1 genewere identified in genetic testing, including 2 known pathogenic variations (c.2359C>T and c.1549+1G>T) and 8 unreported variations (c.3405-1G>T, c.2372C>T, c.1436C>T, c.2228T>C, c.2441G>A, c.3031G>A, c.3075T>C and c.390-403del). All patients had decreased citrulline levels (2.72-6.21 µmol/L), and varying degrees of elevated blood ammonia. The patients received restricted natural protein intake (special formula), arginine and supportive therapy after diagnosis, and were followed-up for a period ranging from 9 months to 10 years. Three patients experienced hyperammonemia, and one patient each had attention deficit hyperactivity disorder, transient facial twitching and increased muscle tone. One patient died, while the other five surviving patients had normal scores of the Ages & Stages Questionnaires (ASQ) and Griffiths Development Scales up to the present time; 4 cases had combined height or weight lag and one case was normal in height and weight. CONCLUSIONS: Low citrulline levels and hyperammonemia are common in CPS1 deficiency patients in Zhejiang. Most gene variants identified were specific to individual families, and no hotspot mutations were found. Early diagnosis through newborn screening and following standardized treatment can significantly improve the prognosis of the patients.
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Enfermedad por Deficiencia de Carbamoil-Fosfato Sintasa I , Hiperamonemia , Niño , Humanos , Recién Nacido , Enfermedad por Deficiencia de Carbamoil-Fosfato Sintasa I/diagnóstico , Enfermedad por Deficiencia de Carbamoil-Fosfato Sintasa I/genética , Enfermedad por Deficiencia de Carbamoil-Fosfato Sintasa I/terapia , Tamizaje Neonatal , Estudios de Seguimiento , Citrulina/genética , Estudios Retrospectivos , MutaciónRESUMEN
Protein citrullination is a post-translational modification (PTM) that is catalysed by the protein arginine deiminase (PAD) family of enzymes. This PTM involves the transformation of an arginine residue into citrulline. Protein citrullination is associated with several physiological processes, including the epigenetic regulation of gene expression, neutrophil extracellular trap formation and DNA damage-induced apoptosis. Aberrant protein citrullination is relevant to several autoimmune and neurodegenerative diseases and certain forms of cancer. PAD inhibitors have shown remarkable efficacy in a range of diseases including rheumatoid arthritis (RA), lupus, atherosclerosis and ulcerative colitis. In RA, anti-citrullinated protein antibodies can be detected prior to disease onset and are thus a valuable diagnostic tool for RA. Notably, citrullinated proteins may serve more generally as biomarkers of specific disease states; however, the identification of citrullinated protein residues remains challenging owing to the small 1 Da mass change that occurs upon citrullination. Herein, we highlight the progress made so far in the development of pan-PAD and isozyme selective inhibitors as well as the identification of citrullinated proteins and the site-specific incorporation of citrulline into proteins. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.
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Artritis Reumatoide , Citrulinación , Humanos , Citrulina/genética , Citrulina/metabolismo , Epigénesis Genética , Proteínas/genética , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/metabolismo , Procesamiento Proteico-PostraduccionalAsunto(s)
Artritis Reumatoide , Citrulina , Humanos , Citrulina/genética , Artritis Reumatoide/genética , Autoanticuerpos , Fenotipo , Péptidos CíclicosRESUMEN
OBJECTIVES@#To investigate genotype-phenotype characteristics and long-term prognosis of neonatal carbamoyl phosphate synthetase 1 (CPS1) deficiency among children through newborn screening in Zhejiang province.@*METHODS@#The clinical and follow-up data of children with CPS1 deficiency detected through neonatal screening and confirmed by tandem mass spectrometry and genetic testing in Zhejiang Province Newborn Disease Screening Center from September 2013 to August 2023 were retrospectively analyzed.@*RESULTS@#A total of 4 056 755 newborns were screened and 6 cases of CPS1 deficiency were diagnosed through phenotypic and genetic testing. Ten different variations of CPS1 genewere identified in genetic testing, including 2 known pathogenic variations (c.2359C>T and c.1549+1G>T) and 8 unreported variations (c.3405-1G>T, c.2372C>T, c.1436C>T, c.2228T>C, c.2441G>A, c.3031G>A, c.3075T>C and c.390-403del). All patients had decreased citrulline levels (2.72-6.21 μmol/L), and varying degrees of elevated blood ammonia. The patients received restricted natural protein intake (special formula), arginine and supportive therapy after diagnosis, and were followed-up for a period ranging from 9 months to 10 years. Three patients experienced hyperammonemia, and one patient each had attention deficit hyperactivity disorder, transient facial twitching and increased muscle tone. One patient died, while the other five surviving patients had normal scores of the Ages & Stages Questionnaires (ASQ) and Griffiths Development Scales up to the present time; 4 cases had combined height or weight lag and one case was normal in height and weight.@*CONCLUSIONS@#Low citrulline levels and hyperammonemia are common in CPS1 deficiency patients in Zhejiang. Most gene variants identified were specific to individual families, and no hotspot mutations were found. Early diagnosis through newborn screening and following standardized treatment can significantly improve the prognosis of the patients.
Asunto(s)
Niño , Humanos , Recién Nacido , Enfermedad por Deficiencia de Carbamoil-Fosfato Sintasa I/terapia , Tamizaje Neonatal , Estudios de Seguimiento , Hiperamonemia , Citrulina/genética , Estudios Retrospectivos , MutaciónRESUMEN
Peptidylarginine deiminase 4 (PAD4), a Ca2+-dependent enzyme, catalyzes the conversion of arginine to citrulline and has been strongly associated with many malignant tumors. However, the molecular mechanisms of PAD4 in the development and progression of colorectal cancer (CRC) remain unclearly defined. In our study, PAD4 expression was increased in CRC tissues and cells, and was closely related to tumor size, lymph node metastasis. Moreover, the transcription factor KLF9 directly bound to PADI4 gene promoter, leading to overexpression of PAD4 in CRC cells, which augmented cell growth and migration. We revealed that PAD4 interacted with and citrullinated glycogen synthase kinase-3ß (GSK3ß) in CRC cells, and GSK3ß Arg-344 was the dominating PAD4-citrullination site. Furthermore, IgL2 and catalytic domains of PAD4 directly bound to the kinase domain of GSK3ß in CRC cells. Mechanistically, PAD4 promoted the transport of GSK3ß from the cytoplasm to the nucleus, thereby increasing the ubiquitin-dependent proteasome degradation of nuclear cyclin-dependent kinase inhibitor 1 (CDKN1A). Our study is the first to reveal the details of a critical PAD4/GSK3ß/CDKN1A signaling axis for CRC progression, and provides evidence that PAD4 is a potential diagnosis biomarker and therapeutic target in CRC.
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Citrulinación , Neoplasias Colorrectales , Arginina/genética , Biomarcadores/metabolismo , Citrulina/genética , Citrulina/metabolismo , Neoplasias Colorrectales/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Hidrolasas/genética , Hidrolasas/metabolismo , Factores de Transcripción de Tipo Kruppel/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Arginina Deiminasa Proteína-Tipo 4 , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/metabolismo , Factores de Transcripción/genética , Ubiquitinas/genéticaRESUMEN
Citrullination, the post-translational modification of arginine residues, is catalyzed by the four catalytically active peptidylarginine deiminase (PAD or PADI) isozymes and alters charge to affect target protein structure and function. PADs were initially characterized in rodent uteri and, since then, have been described in other female tissues including ovaries, breast, and the lactotrope and gonadotrope cells of the anterior pituitary gland. In these tissues and cells, estrogen robustly stimulates PAD expression resulting in changes in levels over the course of the female reproductive cycle. The best-characterized targets for PADs are arginine residues in histone tails, which, when citrullinated, alter chromatin structure and gene expression. Methodological advances have allowed for the identification of tissue-specific citrullinomes, which reveal that PADs citrullinate a wide range of enzymes and structural proteins to alter cell function. In contrast to their important physiological roles, PADs and citrullinated proteins are also involved in several female-specific diseases including autoimmune disorders and reproductive cancers. Herein, we review current knowledge regarding PAD expression and function and highlight the role of protein citrullination in both normal female reproductive tissues and associated diseases.
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Citrulinación , Citrulina , Femenino , Animales , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/metabolismo , Citrulina/genética , Citrulina/metabolismo , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Hidrolasas/genética , Arginina/metabolismoRESUMEN
INTRODUCTION: Citrullinemia Type 1 (CTLN1) is an autosomal recessive disorder caused by variants in the ASS1 gene. This study intends to clarify the etiology of false positives in newborn screening for citrullinemia. METHOD: Newborns who had elevated dried-blood spot citrulline levels were enrolled, and medical records were reviewed retrospectively. Common ASS1 variants were screened using high-resolution melting analysis. RESULT: Between 2011 and 2021, 130 newborns received confirmatory testing for citrullinemia, 4 were found to be patients for CTLN1; 11 were patients with citrin deficiency; and 49 newborns were confirmed to be carrying one pathogenic ASS1 variant. The incidence of CTLN1 was 1 in 188,380 (95% confidence interval: 1 in 73,258 to 1 in 484,416). All ASS1 variants studied in this cohort were located in exons 11 to 15, which encode the tetrameric interface regions of the ASS1 protein. Among 10 ASS1 carriers with elevated citrulline levels and complete sequence data, four (40%) revealed additional non-benign ASS1 variants; in contrast, only 2 of the 26 controls (7.7%), with normal citrulline levels, had additional ASS1 variants. CONCLUSION: Heterozygote ASS1 variants may lead to a mild elevation of blood citrulline levels: about 2-6 times the population mean. Molecular testing and family studies remain critical for precise diagnosis, genetic counseling, and management.
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Citrulinemia , Argininosuccinato Sintasa/genética , Argininosuccinato Sintasa/metabolismo , Citrulina/genética , Citrulina/metabolismo , Citrulinemia/diagnóstico , Citrulinemia/genética , Heterocigoto , Humanos , Recién Nacido , Estudios RetrospectivosRESUMEN
BACKGROUND: Ornithine transcarbamylase deficiency (OTCD) is most common among urea cycle disorders (UCDs), defined by defects in enzymes associated with ureagenesis. Corticosteroid administration to UCD patients, including OTCD patients, is suggested to be avoided, as it may induce life-threatening hyperammonemia. The mechanism has been considered nitrogen overload due to the catabolic effect of corticosteroids; however, the pathophysiological process is unclear. METHODS: To elucidate the mechanism of hyperammonemia induced by corticosteroid administration in OTCD patients, we analyzed a mouse model by administering corticosteroids to OTCspf-ash mice deficient in the OTC gene. Dexamethasone (DEX; 20 mg/kg) was administered to the OTCspf-ash and wild-type (WT) mice at 0 and 24 h, and the serum ammonia concentrations, the levels of the hepatic metabolites, and the gene expressions related with ammonia metabolism in the livers and muscles were analyzed. RESULTS: The ammonia levels in Otcspf-ash mice that were administered DEX tended to increase at 24 h and increased significantly at 48 h. The metabolomic analysis showed that the levels of citrulline, arginine, and ornithine did not differ significantly between Otcspf-ash mice that were administered DEX and normal saline; however, the level of aspartate was increased drastically in Otcspf-ash mice owing to DEX administration (P < 0.01). Among the enzymes associated with the urea cycle, mRNA expressions of carbamoyl-phosphate synthase 1, ornithine transcarbamylase, arginosuccinate synthase 1, and arginosuccinate lyase in the livers were significantly downregulated by DEX administration in both the Otcspf-ash and WT mice (P < 0.01). Among the enzymes associated with catabolism, mRNA expression of Muscle RING-finger protein-1 in the muscles was significantly upregulated in the muscles of WT mice by DEX administration (P < 0.05). CONCLUSIONS: We elucidated that corticosteroid administration induced hyperammonemia in Otcspf-ash mice by not only muscle catabolism but also suppressing urea-cycle-related gene expressions. Since the urea cycle intermediate amino acids, such as arginine, might not be effective because of the suppressed expression of urea-cycle-related genes by corticosteroid administration, we should consider an early intervention by renal replacement therapy in cases of UCD patients induced by corticosteroids to avoid brain injuries or fatal outcomes.
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Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa , Corticoesteroides , Animales , Citrulina/genética , Expresión Génica , Humanos , Ratones , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/genética , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/metabolismo , Urea/metabolismoRESUMEN
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.
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Citrulinación/fisiología , Minería de Datos/métodos , Proteómica/métodos , Algoritmos , Arginina/metabolismo , Citrulinación/genética , Citrulina/química , Citrulina/genética , Citrulina/metabolismo , Análisis de Datos , Manejo de Datos/métodos , Humanos , Péptidos/metabolismo , Procesamiento Proteico-Postraduccional , Desiminasas de la Arginina Proteica/genética , Desiminasas de la Arginina Proteica/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Protein arginine deiminases (PADs), is a group of calcium-dependent enzymes, which play crucial roles in citrullination, and can catalyze arginine residues into citrulline. This chemical reaction induces citrullinated proteins formation with altered structure and function, leading to numerous pathological diseases, including inflammation and autoimmune diseases. To date, multiple studies have provided solid evidence that PADs are implicated in cancer progression. Nevertheless, the findings on PADs functions in tumors are too complex to understand due to its involvements in variable signaling pathways. The increasing interest in PADs has heightened the need for a comprehensive description for its role in cancer. The present study aims to identify the gaps in present knowledge, including its structures, biological substrates and tissue distribution. Since several irreversible inhibitors for PADs with good potency and selectivity have been explored, the mechanisms on the dysregulation in tumors remain poorly understood. The present study discusses the relationship between PADs and tumor apoptosis, EMT formation and metastasis as well as the implication of neutrophil extracellular traps (NETs) in tumorigenesis. In addition, the potential uses of citrullinated antigens for immunotherapy were proposed.
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Citrulinación/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias/genética , Desiminasas de la Arginina Proteica/genética , Citrulina/genética , Citrulina/metabolismo , Trampas Extracelulares/genética , Humanos , Neoplasias/patología , Transducción de Señal/genéticaRESUMEN
Watermelon fruit contains a high percentage of amino acid citrulline (Cit) and arginine (Arg). Cit and Arg accumulation in watermelon fruit are most likely mediated by both de novo synthesis from other amino acids within fruits and direct import from source tissues (leaves) through the phloem. The amino acid transporters involved in the import of Cit, Arg, and their precursors into developing fruits of watermelon have not been reported. In this study, we have compiled the list of putative amino acid transporters in watermelon and characterized transporters that are expressed in the early stage of fruit development. Using the yeast complementation study, we characterized ClAAP3 (Cla023187) and ClAAP6 (Cla023090) as functional amino acid transporters belonging to the family of amino acid permease (AAP) genes. The yeast growth and uptake assays of radiolabeled amino acid suggested that ClAAP3 and ClAAP6 can transport a broad spectrum of amino acids. Expression of translational fusion proteins with a GFP reporter in Nicotiana benthamiana leaves confirmed the ER- and plasma membrane-specific localization, suggesting the role of ClAAP proteins in the cellular import of amino acids. Based on the gene expression profiles and functional characterization, ClAAP3 and ClAAP6 are expected to play a major role in regulation of amino acid import into developing watermelon fruits.
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Sistemas de Transporte de Aminoácidos/biosíntesis , Citrullus/metabolismo , Frutas/metabolismo , Proteínas de Plantas/biosíntesis , Sistemas de Transporte de Aminoácidos/genética , Arginina/genética , Arginina/metabolismo , Citrulina/genética , Citrulina/metabolismo , Citrullus/genética , Frutas/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Especificidad por Sustrato , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
Watermelon is a good source of citrulline, a non-protein amino acid. Citrulline has several therapeutic and clinical implications as it produces nitric oxide via arginine. In plants, citrulline plays a pivotal role in nitrogen transport and osmoprotection. The purpose of this study was to identify single nucleotide polymorphism (SNP) markers associated with citrulline metabolism using a genome-wide association study (GWAS) and understand the role of citrulline in watermelon domestication. A watermelon collection consisting of 187 wild, landraces, and cultivated accessions was used to estimate citrulline content. An association analysis involved a total of 12,125 SNPs with a minor allele frequency (MAF) >0.05 in understanding the population structure and phylogeny in light of citrulline accumulation. Wild egusi types and landraces contained low to medium citrulline content, whereas cultivars had higher content, which suggests that obtaining higher content of citrulline is a domesticated trait. GWAS analysis identified candidate genes (ferrochelatase and acetolactate synthase) showing a significant association of SNPs with citrulline content. Haplotype networking indicated positive selection from wild to domesticated watermelon. To our knowledge, this is the first study showing genetic regulation of citrulline variation in plants by using a GWAS strategy. These results provide new insights into the citrulline metabolism in plants and the possibility of incorporating high citrulline as a trait in watermelon breeding programs.
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Citrulina/genética , Citrulina/metabolismo , Citrullus/genética , Citrullus/metabolismo , Domesticación , Estudio de Asociación del Genoma Completo/métodos , Haplotipos/genética , Acetolactato Sintasa/genética , Acetolactato Sintasa/metabolismo , Arginina , Ferroquelatasa/genética , Ferroquelatasa/metabolismo , Frecuencia de los Genes , Ontología de Genes , Genes de Plantas/genética , Genoma de Planta , Óxido Nítrico , Osmorregulación , Fenotipo , Filogenia , Polimorfismo de Nucleótido SimpleRESUMEN
Pathogenic variants in the argininosuccinate lyase (ASL) gene have been shown to cause argininosuccinate lyase deficiency (ASLD); therefore, sequencing analysis offers advantages for prenatal testing and counseling in families afflicted with this condition. Here, we performed a genetic analysis of an ASLD patient and his family with an aim to offer available information for clinical diagnosis. The research subjects were a 23-month-old patient with a high plasma level of citrulline and his unaffected parents. Whole-exome sequencing identified potential related ASL gene mutations in this trio. Enzymatic activity was detected spectrophotometrically by a coupled assay using arginase and measuring urea production. We identified a novel nonsynonymous mutation (c.206A>G, p.Lys69Arg) and a stop mutation (c.637C>T, p.Arg213∗) in ASL in a Chinese Han patient with ASLD. The enzymatic activity of a p.Lys69Arg ASL construct in human embryonic kidney 293T cells was significantly reduced compared to that of the wild-type construct, and no significant activity was observed for the p.Arg213∗ construct. Compound heterozygous p.Lys69Arg and p.Arg213∗ mutations that resulted in reduced ASL enzyme activity were found in a patient with ASLD. This finding expands the clinical spectrum of ASL pathogenic variants.
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Argininosuccinatoliasa , Aciduria Argininosuccínica , Secuenciación del Exoma , Heterocigoto , Mutación Missense , Sustitución de Aminoácidos , Argininosuccinatoliasa/genética , Argininosuccinatoliasa/metabolismo , Aciduria Argininosuccínica/enzimología , Aciduria Argininosuccínica/genética , Citrulina/sangre , Citrulina/genética , Células HEK293 , Humanos , Lactante , MasculinoRESUMEN
Voltage-dependent activation of voltage-gated cation channels results from the outward movement of arginine-bearing helices within proteinaceous voltage sensors. The voltage-sensing residues in potassium channels have been extensively characterized, but current functional approaches do not allow a distinction between the electrostatic and steric contributions of the arginine side chain. Here we use chemical misacylation and in vivo nonsense suppression to encode citrulline, a neutral and nearly isosteric analogue of arginine, into the voltage sensor of the Shaker potassium channel. We functionally characterize the engineered channels and compare them with those bearing conventional mutations at the same positions. We observe effects on both voltage sensitivity and gating kinetics, enabling dissection of the roles of residue structure versus positive charge in channel function. In some positions, substitution with citrulline causes mild effects on channel activation compared with natural mutations. In contrast, substitution of the fourth S4 arginine with citrulline causes substantial changes in the conductance-voltage relationship and the kinetics of the channel, which suggests that a positive charge is required at this position for efficient voltage sensor deactivation and channel closure. The encoding of citrulline is expected to enable enhanced precision for the study of arginine residues located in crowded transmembrane environments in other membrane proteins. In addition, the method may facilitate the study of citrullination in vivo.
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Arginina/química , Citrulina/química , Activación del Canal Iónico , Canales de Potasio de la Superfamilia Shaker/química , Sustitución de Aminoácidos , Animales , Arginina/genética , Citrulina/genética , Potenciales de la Membrana , Ratones , Dominios Proteicos , Canales de Potasio de la Superfamilia Shaker/genética , Canales de Potasio de la Superfamilia Shaker/metabolismo , Electricidad Estática , XenopusRESUMEN
Pentraxins are fluid phase pattern recognition molecules that form an important part of the innate immune defence and are conserved between fish and human. In Atlantic cod (Gadus morhua L.), two pentraxin-like proteins have been described, CRP-I and CRP-II. Here we show for the first time that these two CRP forms are post-translationally deiminated (an irreversible conversion of arginine to citrulline) and differ with respect to tissue specific localisation in cod ontogeny from 3 to 84 days post hatching. While both forms are expressed in liver, albeit at temporally differing levels, CRP-I shows a strong association with nervous tissue while CRP-II is strongly associated to mucosal tissues of gut and skin. This indicates differing roles for the two pentraxin types in immune responses and tissue remodelling, also elucidating novel roles for CRP-I in the nervous system. The presence of deimination positive bands for cod CRPs varied somewhat between mucus and serum, possibly facilitating CRP protein moonlighting, allowing the same protein to exhibit a range of biological functions and thus meeting different functional requirements in different tissues. The presented findings may further current understanding of the diverse roles of pentraxins in teleost immune defences and tissue remodelling, as well as in various human pathologies, including autoimmune diseases, amyloidosis and cancer.
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Proteína C-Reactiva/inmunología , Proteínas de Peces/inmunología , Gadus morhua/inmunología , Animales , Arginina/genética , Arginina/inmunología , Arginina/metabolismo , Proteína C-Reactiva/genética , Proteína C-Reactiva/metabolismo , Citrulina/genética , Citrulina/inmunología , Citrulina/metabolismo , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Gadus morhua/genética , Gadus morhua/metabolismo , Humanos , Membrana Mucosa/inmunología , Membrana Mucosa/metabolismo , Tejido Nervioso/inmunología , Tejido Nervioso/metabolismo , Especificidad de Órganos/genética , Especificidad de Órganos/inmunología , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional/inmunologíaRESUMEN
Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.
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Cianatos , Cianuros , Peroxidasa , Placa Aterosclerótica/enzimología , Carbamilación de Proteína , Animales , Citrulina/análogos & derivados , Citrulina/química , Citrulina/genética , Citrulina/metabolismo , Cianatos/química , Cianatos/metabolismo , Cianuros/química , Cianuros/metabolismo , Humanos , Ratones , Ratones Noqueados , Oxidación-Reducción , Peroxidasa/química , Peroxidasa/genética , Peroxidasa/metabolismo , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologíaRESUMEN
OBJECTIVE: The pathogenetic mechanisms by which HLA-DRB1 alleles are associated with anticitrullinated peptide antibody (ACPA)-positive rheumatoid arthritis (RA) are incompletely understood. RA high-risk HLA-DRB1 alleles are known to share a common motif, the 'shared susceptibility epitope (SE)'. Here, the electropositive P4 pocket of HLA-DRB1 accommodates self-peptide residues containing citrulline but not arginine. HLA-DRB1 His/Phe13ß stratifies with ACPA-positive RA, while His13ßSer polymorphisms stratify with ACPA-negative RA and RA protection. Indigenous North American (INA) populations have high risk of early-onset ACPA-positive RA, whereby HLA-DRB1*04:04 and HLA-DRB1*14:02 are implicated as risk factors for RA in INA. However, HLA-DRB1*14:02 has a His13ßSer polymorphism. Therefore, we aimed to verify this association and determine its molecular mechanism. METHODS: HLA genotype was compared in 344 INA patients with RA and 352 controls. Structures of HLA-DRB1*1402-class II loaded with vimentin-64Arg59-71, vimentin-64Cit59-71 and fibrinogen ß-74Cit69-81 were solved using X-ray crystallography. Vimentin-64Cit59-71-specific and vimentin59-71-specific CD4+ T cells were characterised by flow cytometry using peptide-histocompatibility leukocyte antigen (pHLA) tetramers. After sorting of antigen-specific T cells, TCRα and ß-chains were analysed using multiplex, nested PCR and sequencing. RESULTS: ACPA+ RA in INA was independently associated with HLA-DRB1*14:02. Consequent to the His13ßSer polymorphism and altered P4 pocket of HLA-DRB1*14:02, both citrulline and arginine were accommodated in opposite orientations. Oligoclonal autoreactive CD4+ effector T cells reactive with both citrulline and arginine forms of vimentin59-71 were observed in patients with HLA-DRB1*14:02+ RA and at-risk ACPA- first-degree relatives. HLA-DRB1*14:02-vimentin59-71-specific and HLA-DRB1*14:02-vimentin-64Cit59-71-specific CD4+ memory T cells were phenotypically distinct populations. CONCLUSION: HLA-DRB1*14:02 broadens the capacity for citrullinated and native self-peptide presentation and T cell expansion, increasing risk of ACPA+ RA.
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/genética , Artritis Reumatoide/etnología , Artritis Reumatoide/genética , Predisposición Genética a la Enfermedad/etnología , Cadenas HLA-DRB1/genética , Indígenas Norteamericanos/genética , Alaska/etnología , Alelos , Arginina/genética , Arginina/inmunología , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Linfocitos T CD4-Positivos/inmunología , Canadá/etnología , Estudios de Casos y Controles , Citrulina/genética , Citrulina/inmunología , Femenino , Citometría de Flujo , Genotipo , Humanos , Masculino , Péptidos Cíclicos/inmunología , Polimorfismo Genético , Factores de Riesgo , Vimentina/genéticaRESUMEN
Citrulline was chemically isolated more than 100 years ago and is ubiquitous in animals, plants, bacteria, and fungi. Most of the research on plant citrulline metabolism and transport has been carried out in Arabidopsis thaliana and the Cucurbitaceae family, particularly in watermelon which accumulates this non-proteinogenic amino acid to very high levels. Industrially, citrulline is produced via specially optimized microbial strains; however, the amounts present in watermelon render it an economically viable source providing that other high-value compounds can be co-extracted. In this review, we provide an overview of our current understanding of citrulline biosynthesis, transport, and catabolism in plants additionally pointing out significant gaps in our knowledge which need to be closed by future experimentation. This includes the identification of further potential enzymes of citrulline metabolism as well as obtaining a far better spatial resolution of both sub-cellular and long-distance partitioning of citrulline. We further discuss what is known concerning the biological function of citrulline in plants paying particular attention to the proposed roles in scavenging of excess NH4+ and as a compatible solute.
Asunto(s)
Arabidopsis/metabolismo , Citrulina/biosíntesis , Cucurbitaceae/metabolismo , Regulación de la Expresión Génica de las Plantas , Ornitina Carbamoiltransferasa/genética , Adaptación Fisiológica , Sistemas de Transporte de Aminoácidos/genética , Sistemas de Transporte de Aminoácidos/metabolismo , Amoníaco/metabolismo , Animales , Arabidopsis/genética , Argininosuccinato Sintasa/genética , Argininosuccinato Sintasa/metabolismo , Bacterias/metabolismo , Transporte Biológico , Carbamoil Fosfato/metabolismo , Citrulina/genética , Cucurbitaceae/genética , Hongos/metabolismo , Humanos , Ornitina/metabolismo , Ornitina Carbamoiltransferasa/metabolismo , Estrés FisiológicoRESUMEN
Despite the absence of any homologs of Tannerella forsythia KLIKK proteases in Tannerella sp.6_1_58FAA_CT1, the strain possesses a putative cysteine protease (G9S4N1) closely related to RgpB of Porphyromonas gingivalis. G9S4N1 lacks obvious propeptide that behaves as inhibitor of proteases and was proven to be a propeptide-independent protease. Unlike RgpB, which exclusively cleaves ArgXaa bonds, G9S4N1 exhibits both arginine- and citrulline-specific activities. Mutations of Asp177, a potential P1-Arg binding site, to uncharged or positively charged residues did not alter the substrate specificity of G9S4N1 significantly. Moreover, a group of arginine-specific proteases from different species including porcine trypsin, bovine thrombin, and a trypsin-like serine protease of dengue 2 virus CF40-Gly-NS3pro185 also display different specificity toward citrulline residue, suggesting that citrulline-modified protein might have different roles and destiny in biological processes involving various proteases.
Asunto(s)
Bacteroidetes/enzimología , Proteasas de Cisteína/metabolismo , Adhesinas Bacterianas/genética , Animales , Arginina/metabolismo , Bacteroidetes/metabolismo , Sitios de Unión , Bovinos , Citrulina/genética , Citrulina/metabolismo , Cisteína Endopeptidasas/genética , Proteasas de Cisteína/química , Proteasas de Cisteína/genética , Cisteína-Endopeptidasas Gingipaínas , Cinética , Lisina/metabolismo , Modelos Moleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Porcinos , Tannerella forsythia , Tripsina/metabolismoRESUMEN
Cathelicidin LL-37 plays an essential role in innate immunity by killing invading microorganisms and regulating the inflammatory response. These activities depend on the cationic character of the peptide, which is conferred by arginine and lysine residues. At inflammatory foci in vivo, LL-37 is exposed to peptidyl arginine deiminase (PAD), an enzyme released by inflammatory cells. Therefore, we hypothesized that PAD-mediated citrullination of the arginine residues within LL-37 will abrogate its immunomodulatory functions. We found that, when citrullinated, LL-37 was at least 40 times less efficient at neutralizing the proinflammatory activity of LPS due to a marked decrease in its affinity for endotoxin. Also, the ability of citrullinated LL-37 to quench macrophage responses to lipoteichoic acid and poly(I:C) signaling via TLR2 and TLR3, respectively, was significantly reduced. Furthermore, in stark contrast to native LL-37, the modified peptide completely lost the ability to prevent morbidity and mortality in a mouse model of d-galactosamine-sensitized endotoxin shock. In fact, administration of citrullinated LL-37 plus endotoxin actually exacerbated sepsis due to the inability of LL-37 to neutralize LPS and the subsequent enhancement of systemic inflammation due to increased serum levels of IL-6. Importantly, serum from septic mice showed increased PAD activity, which strongly correlated with the level of citrullination, indicating that PAD-driven protein modification occurs in vivo. Because LL-37 is a potential treatment for sepsis, its administration should be preceded by a careful analysis to ensure that the citrullinated peptide is not generated in treated patients.