RESUMO
The intimate relationship between the epithelium and immune system is crucial for maintaining tissue homeostasis, with perturbations therein linked to autoimmune disease and cancer1-3. Whereas stem cell-derived organoids are powerful models of epithelial function4, they lack tissue-resident immune cells that are essential for capturing organ-level processes. We describe human intestinal immuno-organoids (IIOs), formed through self-organization of epithelial organoids and autologous tissue-resident memory T (TRM) cells, a portion of which integrate within the epithelium and continuously survey the barrier. TRM cell migration and interaction with epithelial cells was orchestrated by TRM cell-enriched transcriptomic programs governing cell motility and adhesion. We combined IIOs and single-cell transcriptomics to investigate intestinal inflammation triggered by cancer-targeting biologics in patients. Inflammation was associated with the emergence of an activated population of CD8+ T cells that progressively acquired intraepithelial and cytotoxic features. The appearance of this effector population was preceded and potentiated by a T helper-1-like CD4+ population, which initially produced cytokines and subsequently became cytotoxic itself. As a system amenable to direct perturbation, IIOs allowed us to identify the Rho pathway as a new target for mitigation of immunotherapy-associated intestinal inflammation. Given that they recapitulate both the phenotypic outcomes and underlying interlineage immune interactions, IIOs can be used to study tissue-resident immune responses in the context of tumorigenesis and infectious and autoimmune diseases.
Assuntos
Linfócitos T CD8-Positivos , Células T de Memória , Organoides , Organoides/imunologia , Humanos , Células T de Memória/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/citologia , Análise de Célula Única , Movimento Celular/imunologia , Inflamação/imunologia , Inflamação/patologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/citologia , Intestinos/imunologia , Intestinos/citologia , Transcriptoma , Células Epiteliais/imunologia , Células Epiteliais/citologia , Feminino , Imunoterapia , Masculino , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/citologiaRESUMO
Influenza A virus exhibits high rates of replicative failure due to a variety of genetic defects. Most influenza virions cannot, when acting as individual particles, complete the entire viral life cycle. Nevertheless influenza is incredibly successful in the suppression of innate immune detection and the production of interferons, remaining undetected in >99% of cells in tissue-culture models of infection. Notably, the same variation that leads to replication failure can, by chance, inactivate the major innate immune antagonist in influenza A virus, NS1. What explains the observed rarity of interferon production in spite of the frequent loss of this, critical, antagonist? By studying how genetic and phenotypic variation in a viral population lacking NS1 correlates with interferon production, we have built a model of the "worst-case" failure from an improved understanding of the steps at which NS1 acts in the viral life cycle to prevent the triggering of an innate immune response. In doing so, we find that NS1 prevents the detection of de novo innate immune ligands, defective viral genomes, and viral export from the nucleus, although only generation of de novo ligands appears absolutely required for enhanced detection of virus in the absence of NS1. Due to this, the highest frequency of interferon production we observe (97% of infected cells) requires a high level of replication in the presence of defective viral genomes with NS1 bearing an inactivating mutation that does not impact its partner encoded on the same segment, NEP. This is incredibly unlikely to occur given the standard variation found within a viral population, and would generally require direct, artificial, intervention to achieve at an appreciable rate. Thus from our study, we procure at least a partial explanation for the seeming contradiction between high rates of replicative failure and the rarity of the interferon response to influenza infection.
Assuntos
Vírus da Influenza A , Influenza Humana , Humanos , Interferons/genética , Influenza Humana/genética , Proteínas não Estruturais Virais/genética , Vírus da Influenza A/genética , Imunidade Inata , Replicação Viral/genéticaRESUMO
The vasculature is a key regulator of leukocyte trafficking into the central nervous system (CNS) during inflammatory diseases including multiple sclerosis (MS). However, the impact of endothelial-derived factors on CNS immune responses remains unknown. Bioactive lipids, in particular oxysterols downstream of Cholesterol-25-hydroxylase (Ch25h), promote neuroinflammation but their functions in the CNS are not well-understood. Using floxed-reporter Ch25h knock-in mice, we trace Ch25h expression to CNS endothelial cells (ECs) and myeloid cells and demonstrate that Ch25h ablation specifically from ECs attenuates experimental autoimmune encephalomyelitis (EAE). Mechanistically, inflamed Ch25h-deficient CNS ECs display altered lipid metabolism favoring polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) expansion, which suppresses encephalitogenic T lymphocyte proliferation. Additionally, endothelial Ch25h-deficiency combined with immature neutrophil mobilization into the blood circulation nearly completely protects mice from EAE. Our findings reveal a central role for CNS endothelial Ch25h in promoting neuroinflammation by inhibiting the expansion of immunosuppressive myeloid cell populations.
Assuntos
Encefalomielite Autoimune Experimental , Oxisteróis , Camundongos , Animais , Células Endoteliais/metabolismo , Oxisteróis/metabolismo , Doenças Neuroinflamatórias , Sistema Nervoso Central/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Trichothiodystrophy (TTD) is a rare hereditary neurodevelopmental disorder defined by sulfur-deficient brittle hair and nails and scaly skin, but with otherwise remarkably variable clinical features. The photosensitive TTD (PS-TTD) forms exhibits in addition to progressive neuropathy and other features of segmental accelerated aging and is associated with impaired genome maintenance and transcription. New factors involved in various steps of gene expression have been identified for the different non-photosensitive forms of TTD (NPS-TTD), which do not appear to show features of premature aging. Here, we identify alanyl-tRNA synthetase 1 and methionyl-tRNA synthetase 1 variants as new gene defects that cause NPS-TTD. These variants result in the instability of the respective gene products alanyl- and methionyl-tRNA synthetase. These findings extend our previous observations that TTD mutations affect the stability of the corresponding proteins and emphasize this phenomenon as a common feature of TTD. Functional studies in skin fibroblasts from affected individuals demonstrate that these new variants also impact on the rate of tRNA charging, which is the first step in protein translation. The extension of reduced abundance of TTD factors to translation as well as transcription redefines TTD as a syndrome in which proteins involved in gene expression are unstable.
Assuntos
Alanina-tRNA Ligase/genética , Metionina tRNA Ligase/genética , Síndromes de Tricotiodistrofia/genética , Alanina-tRNA Ligase/metabolismo , Criança , Estabilidade Enzimática/genética , Feminino , Humanos , Metionina tRNA Ligase/metabolismo , Síndromes de Tricotiodistrofia/enzimologia , Síndromes de Tricotiodistrofia/patologia , Sequenciamento Completo do GenomaRESUMO
Aminoacyl-tRNA synthetases (ARSs) are ubiquitous, ancient enzymes that charge amino acids to cognate tRNA molecules, the essential first step of protein translation. Here, we describe 32 individuals from 21 families, presenting with microcephaly, neurodevelopmental delay, seizures, peripheral neuropathy, and ataxia, with de novo heterozygous and bi-allelic mutations in asparaginyl-tRNA synthetase (NARS1). We demonstrate a reduction in NARS1 mRNA expression as well as in NARS1 enzyme levels and activity in both individual fibroblasts and induced neural progenitor cells (iNPCs). Molecular modeling of the recessive c.1633C>T (p.Arg545Cys) variant shows weaker spatial positioning and tRNA selectivity. We conclude that de novo and bi-allelic mutations in NARS1 are a significant cause of neurodevelopmental disease, where the mechanism for de novo variants could be toxic gain-of-function and for recessive variants, partial loss-of-function.
Assuntos
Aspartato-tRNA Ligase/genética , Mutação com Ganho de Função/genética , Mutação com Perda de Função/genética , Transtornos do Neurodesenvolvimento/genética , Aminoacil-RNA de Transferência/genética , Alelos , Aminoacil-tRNA Sintetases/genética , Linhagem Celular , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Linhagem , RNA de Transferência/genética , Células-Tronco/fisiologiaRESUMO
Found in a diverse set of viral populations, defective interfering particles are parasitic variants that are unable to replicate on their own yet rise to relatively high frequencies. Their presence is associated with a loss of population fitness, both through the depletion of key cellular resources and the stimulation of innate immunity. For influenza A virus, these particles contain large internal deletions in the genomic segments which encode components of the heterotrimeric polymerase. Using a library-based approach, we comprehensively profile the growth and replication of defective influenza species, demonstrating that they possess an advantage during genome replication, and that exclusion during population expansion reshapes population composition in a manner consistent with their final, observed, distribution in natural populations. We find that an innate immune response is not linked to the size of a deletion; however, replication of defective segments can enhance their immunostimulatory properties. Overall, our results address several key questions in defective influenza A virus biology, and the methods we have developed to answer those questions may be broadly applied to other defective viruses.
Assuntos
Vírus Defeituosos/genética , Aptidão Genética/genética , Vírus da Influenza A/genética , Animais , Linhagem Celular , Genoma Viral , HumanosRESUMO
Aminoacylation of transfer RNA (tRNA) is a key step in protein biosynthesis, carried out by highly specific aminoacyl-tRNA synthetases (ARSs). ARSs have been implicated in autosomal dominant and autosomal recessive human disorders. Autosomal dominant variants in tryptophanyl-tRNA synthetase 1 (WARS1) are known to cause distal hereditary motor neuropathy and Charcot-Marie-Tooth disease, but a recessively inherited phenotype is yet to be clearly defined. Seryl-tRNA synthetase 1 (SARS1) has rarely been implicated in an autosomal recessive developmental disorder. Here, we report five individuals with biallelic missense variants in WARS1 or SARS1, who presented with an overlapping phenotype of microcephaly, developmental delay, intellectual disability, and brain anomalies. Structural mapping showed that the SARS1 variant is located directly within the enzyme's active site, most likely diminishing activity, while the WARS1 variant is located in the N-terminal domain. We further characterize the identified WARS1 variant by showing that it negatively impacts protein abundance and is unable to rescue the phenotype of a CRISPR/Cas9 wars1 knockout zebrafish model. In summary, we describe two overlapping autosomal recessive syndromes caused by variants in WARS1 and SARS1, present functional insights into the pathogenesis of the WARS1-related syndrome and define an emerging disease spectrum: ARS-related developmental disorders with or without microcephaly.
Assuntos
Aminoacil-tRNA Sintetases , Doença de Charcot-Marie-Tooth , Microcefalia , Triptofano-tRNA Ligase , Animais , Humanos , Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/genética , Ligases , Microcefalia/genética , Microcefalia/patologia , RNA de Transferência , Triptofano-tRNA Ligase/genética , Peixe-Zebra/genéticaRESUMO
Brittle and "tiger-tail" hair is the diagnostic hallmark of trichothiodystrophy (TTD), a rare recessive disease associated with a wide spectrum of clinical features including ichthyosis, intellectual disability, decreased fertility, and short stature. As a result of premature abrogation of terminal differentiation, the hair is brittle and fragile and contains reduced cysteine content. Hypersensitivity to UV light is found in about half of individuals with TTD; all of these individuals harbor bi-allelic mutations in components of the basal transcription factor TFIIH, and these mutations lead to impaired nucleotide excision repair and basal transcription. Different genes have been found to be associated with non-photosensitive TTD (NPS-TTD); these include MPLKIP (also called TTDN1), GTF2E2 (also called TFIIEß), and RNF113A. However, a relatively large group of these individuals with NPS-TTD have remained genetically uncharacterized. Here we present the identification of an NPS-TTD-associated gene, threonyl-tRNA synthetase (TARS), found by next-generation sequencing of a group of uncharacterized individuals with NPS-TTD. One individual has compound heterozygous TARS variants, c.826A>G (p.Lys276Glu) and c.1912C>T (p.Arg638∗), whereas a second individual is homozygous for the TARS variant: c.680T>C (p.Leu227Pro). We showed that these variants have a profound effect on TARS protein stability and enzymatic function. Our results expand the spectrum of genes involved in TTD to include genes implicated in amino acid charging of tRNA, which is required for the last step in gene expression, namely protein translation. We previously proposed that some of the TTD-specific features derive from subtle transcription defects as a consequence of unstable transcription factors. We now extend the definition of TTD from a transcription syndrome to a "gene-expression" syndrome.
Assuntos
Doenças do Cabelo/patologia , Mutação , Treonina-tRNA Ligase/genética , Síndromes de Tricotiodistrofia/patologia , Alelos , Sequência de Aminoácidos , Estudos de Casos e Controles , Doenças do Cabelo/genética , Humanos , Fenótipo , Homologia de Sequência , Fator de Transcrição TFIIH/genética , Síndromes de Tricotiodistrofia/genéticaRESUMO
Aminoacyl-tRNA synthetases (ARSs) are essential enzymes responsible for charging tRNA molecules with cognate amino acids. Consistent with the essential function and ubiquitous expression of ARSs, mutations in 32 of the 37 ARS-encoding loci cause severe, early-onset recessive phenotypes. Previous genetic and functional data suggest a loss-of-function mechanism; however, our understanding of the allelic and locus heterogeneity of ARS-related disease is incomplete. Cysteinyl-tRNA synthetase (CARS) encodes the enzyme that charges tRNACys with cysteine in the cytoplasm. To date, CARS variants have not been implicated in any human disease phenotype. Here, we report on four subjects from three families with complex syndromes that include microcephaly, developmental delay, and brittle hair and nails. Each affected person carries bi-allelic CARS variants: one individual is compound heterozygous for c.1138C>T (p.Gln380∗) and c.1022G>A (p.Arg341His), two related individuals are compound heterozygous for c.1076C>T (p.Ser359Leu) and c.1199T>A (p.Leu400Gln), and one individual is homozygous for c.2061dup (p.Ser688Glnfs∗2). Measurement of protein abundance, yeast complementation assays, and assessments of tRNA charging indicate that each CARS variant causes a loss-of-function effect. Compared to subjects with previously reported ARS-related diseases, individuals with bi-allelic CARS variants are unique in presenting with a brittle-hair-and-nail phenotype, which most likely reflects the high cysteine content in human keratins. In sum, our efforts implicate CARS variants in human inherited disease, expand the locus and clinical heterogeneity of ARS-related clinical phenotypes, and further support impaired tRNA charging as the primary mechanism of recessive ARS-related disease.
Assuntos
Aminoacil-tRNA Sintetases/genética , Doença de Charcot-Marie-Tooth/etiologia , Deficiências do Desenvolvimento/etiologia , Doenças do Cabelo/etiologia , Microcefalia/etiologia , Mutação , Doenças da Unha/etiologia , Adulto , Sequência de Aminoácidos , Doença de Charcot-Marie-Tooth/enzimologia , Doença de Charcot-Marie-Tooth/patologia , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/patologia , Feminino , Genes Recessivos , Predisposição Genética para Doença , Doenças do Cabelo/enzimologia , Doenças do Cabelo/patologia , Humanos , Masculino , Microcefalia/enzimologia , Microcefalia/patologia , Doenças da Unha/enzimologia , Doenças da Unha/patologia , Linhagem , Fenótipo , Prognóstico , Homologia de Sequência , Adulto JovemRESUMO
Aminoacyl-tRNA synthetases (aaRS) are ubiquitously expressed enzymes responsible for ligating amino acids to their cognate tRNA molecules through an aminoacylation reaction. The resulting aminoacyl-tRNA is delivered to ribosome elongation factors to participate in protein synthesis. Seryl-tRNA synthetase (SARS1) is one of the cytosolic aaRSs and catalyzes serine attachment to tRNASer . SARS1 deficiency has already been associated with moderate intellectual disability, ataxia, muscle weakness, and seizure in one family. We describe here a new clinical presentation including developmental delay, central deafness, cardiomyopathy, and metabolic decompensation during fever leading to death, in a consanguineous Turkish family, with biallelic variants (c.638G>T, p.(Arg213Leu)) in SARS1. This missense variant was shown to lead to protein instability, resulting in reduced protein level and enzymatic activity. Our results describe a new clinical entity and expand the clinical and mutational spectrum of SARS1 and aaRS deficiencies.
Assuntos
Aminoacil-tRNA Sintetases , Cardiomiopatias , Surdez , Aminoacil-tRNA Sintetases/genética , Aminoacilação , Cardiomiopatias/genética , Criança , Surdez/genética , Humanos , Perda de HeterozigosidadeRESUMO
Hypomyelinating leukodystrophies are genetic disorders characterized by insufficient myelin deposition during development. They are diagnosed on the basis of both clinical and MRI features followed by genetic confirmation. Here, we report on four unrelated affected individuals with hypomyelination and bi-allelic pathogenic variants in EPRS, the gene encoding cytoplasmic glutamyl-prolyl-aminoacyl-tRNA synthetase. EPRS is a bifunctional aminoacyl-tRNA synthetase that catalyzes the aminoacylation of glutamic acid and proline tRNA species. It is a subunit of a large multisynthetase complex composed of eight aminoacyl-tRNA synthetases and its three interacting proteins. In total, five different EPRS mutations were identified. The p.Pro1115Arg variation did not affect the assembly of the multisynthetase complex (MSC) as monitored by affinity purification-mass spectrometry. However, immunoblot analyses on protein extracts from fibroblasts of the two affected individuals sharing the p.Pro1115Arg variant showed reduced EPRS amounts. EPRS activity was reduced in one affected individual's lymphoblasts and in a purified recombinant protein model. Interestingly, two other cytoplasmic aminoacyl-tRNA synthetases have previously been implicated in hypomyelinating leukodystrophies bearing clinical and radiological similarities to those in the individuals we studied. We therefore hypothesized that leukodystrophies caused by mutations in genes encoding cytoplasmic aminoacyl-tRNA synthetases share a common underlying mechanism, such as reduced protein availability, abnormal assembly of the multisynthetase complex, and/or abnormal aminoacylation, all resulting in reduced translation capacity and insufficient myelin deposition in the developing brain.
Assuntos
Alelos , Aminoacil-tRNA Sintetases/genética , Mutação/genética , Adolescente , Criança , Pré-Escolar , Evolução Fatal , Feminino , Doenças Desmielinizantes Hereditárias do Sistema Nervoso Central , Humanos , Imageamento por Ressonância Magnética , Masculino , Adulto JovemRESUMO
PURPOSE: Recessive cytosolic aminoacyl-tRNA synthetase (ARS) deficiencies are severe multiorgan diseases, with limited treatment options. By loading transfer RNAs (tRNAs) with their cognate amino acids, ARS are essential for protein translation. However, it remains unknown why ARS deficiencies lead to specific symptoms, especially early life and during infections. We set out to increase pathophysiological insight and improve therapeutic possibilities. METHODS: In fibroblasts from patients with isoleucyl-RS (IARS), leucyl-RS (LARS), phenylalanyl-RS-beta-subunit (FARSB), and seryl-RS (SARS) deficiencies, we investigated aminoacylation activity, thermostability, and sensitivity to ARS-specific amino acid concentrations, and developed personalized treatments. RESULTS: Aminoacylation activity was reduced in all patients, and further diminished at 38.5/40 °C (PLARS and PFARSB), consistent with infectious deteriorations. With lower cognate amino acid concentrations, patient fibroblast growth was severely affected. To prevent local and/or temporal deficiencies, we treated patients with corresponding amino acids (follow-up: 1/2-2 2/3rd years), and intensified treatment during infections. All patients showed beneficial treatment effects, most strikingly in growth (without tube feeding), head circumference, development, coping with infections, and oxygen dependency. CONCLUSION: For these four ARS deficiencies, we observed a common disease mechanism of episodic insufficient aminoacylation to meet translational demands and illustrate the power of amino acid supplementation for the expanding ARS patient group. Moreover, we provide a strategy for personalized preclinical functional evaluation.
Assuntos
Aminoacil-tRNA Sintetases , Aminoácidos , Aminoacil-tRNA Sintetases/genética , Aminoacilação , Humanos , RNA de Transferência/metabolismoRESUMO
PURPOSE: Recent reports of individuals with cytoplasmic transfer RNA (tRNA) synthetase-related disorders have identified cases with phenotypic variability from the index presentations. We sought to assess phenotypic variability in individuals with AARS1-related disease. METHODS: A cross-sectional survey was performed on individuals with biallelic variants in AARS1. Clinical data, neuroimaging, and genetic testing results were reviewed. Alanyl tRNA synthetase (AlaRS) activity was measured in available fibroblasts. RESULTS: We identified 11 affected individuals. Two phenotypic presentations emerged, one with early infantile-onset disease resembling the index cases of AARS1-related epileptic encephalopathy with deficient myelination (n = 7). The second (n = 4) was a later-onset disorder, where disease onset occurred after the first year of life and was characterized on neuroimaging by a progressive posterior predominant leukoencephalopathy evolving to include the frontal white matter. AlaRS activity was significantly reduced in five affected individuals with both early infantile-onset and late-onset phenotypes. CONCLUSION: We suggest that variants in AARS1 result in a broader clinical spectrum than previously appreciated. The predominant form results in early infantile-onset disease with epileptic encephalopathy and deficient myelination. However, a subgroup of affected individuals manifests with late-onset disease and similarly rapid progressive clinical decline. Longitudinal imaging and clinical follow-up will be valuable in understanding factors affecting disease progression and outcome.
Assuntos
Leucoencefalopatias , Estudos Transversais , Progressão da Doença , Humanos , Leucoencefalopatias/diagnóstico por imagem , Leucoencefalopatias/genética , FenótipoRESUMO
Aminoacyl-tRNA synthetases (ARSs) catalyze the first step of protein biosynthesis (canonical function) and have additional (non-canonical) functions outside of translation. Bi-allelic pathogenic variants in genes encoding ARSs are associated with various recessive mitochondrial and multisystem disorders. We describe here a multisystem clinical phenotype based on bi-allelic mutations in the two genes (FARSA, FARSB) encoding distinct subunits for tetrameric cytosolic phenylalanyl-tRNA synthetase (FARS1). Interstitial lung disease with cholesterol pneumonitis on histology emerged as an early characteristic feature and significantly determined disease burden. Additional clinical characteristics of the patients included neurological findings, liver dysfunction, and connective tissue, muscular and vascular abnormalities. Structural modeling of newly identified missense mutations in the alpha subunit of FARS1, FARSA, showed exclusive mapping to the enzyme's conserved catalytic domain. Patient-derived mutant cells displayed compromised aminoacylation activity in two cases, while remaining unaffected in another. Collectively, these findings expand current knowledge about the human ARS disease spectrum and support a loss of canonical and non-canonical function in FARS1-associated recessive disease.
Assuntos
Doença de Charcot-Marie-Tooth/genética , Doenças Pulmonares Intersticiais/genética , Pulmão/patologia , Mutação/genética , Fenilalanina-tRNA Ligase/genética , Adolescente , Alelos , Criança , Pré-Escolar , Feminino , Genes Recessivos/genética , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , FenótipoRESUMO
PURPOSE: Biallelic variants in LARS1, coding for the cytosolic leucyl-tRNA synthetase, cause infantile liver failure syndrome 1 (ILFS1). Since its description in 2012, there has been no systematic analysis of the clinical spectrum and genetic findings. METHODS: Individuals with biallelic variants in LARS1 were included through an international, multicenter collaboration including novel and previously published patients. Clinical variables were analyzed and functional studies were performed in patient-derived fibroblasts. RESULTS: Twenty-five individuals from 15 families were ascertained including 12 novel patients with eight previously unreported variants. The most prominent clinical findings are recurrent elevation of liver transaminases up to liver failure and encephalopathic episodes, both triggered by febrile illness. Magnetic resonance image (MRI) changes during an encephalopathic episode can be consistent with metabolic stroke. Furthermore, growth retardation, microcytic anemia, neurodevelopmental delay, muscular hypotonia, and infection-related seizures are prevalent. Aminoacylation activity is significantly decreased in all patient cells studied upon temperature elevation in vitro. CONCLUSION: ILFS1 is characterized by recurrent elevation of liver transaminases up to liver failure in conjunction with abnormalities of growth, blood, nervous system, and musculature. Encephalopathic episodes with seizures can occur independently from liver crises and may present with metabolic stroke.
Assuntos
Falência Hepática , Humanos , Hipotonia Muscular , Mutação , ConvulsõesRESUMO
We describe 14 patients with 12 novel missense mutations in ASPA, the gene causing Canavan disease (CD). We developed a method to study the effect of these 12 variants on the function of aspartoacylase-the hydrolysis of N-acetyl-l-aspartic acid (NAA) to aspartate and acetate. The wild-type ASPA open reading frame (ORF) and the ORFs containing each of the variants were transfected into HEK293 cells. Enzyme activity was determined by incubating cell lysates with NAA and measuring the released aspartic acid by LC-MS/MS. Clinical data were obtained for 11 patients by means of questionnaires. Four patients presented with a non-typical clinical picture or with the milder form of CD, whereas seven presented with severe CD. The mutations found in the mild patients corresponded to the variants with the highest residual enzyme activities, suggesting that this assay can help evaluate unknown variants found in patients with atypical presentation. We have detected a correlation between clinical presentation, enzyme activity, and genotype for CD.
Assuntos
Amidoidrolases/metabolismo , Doença de Canavan/diagnóstico , Doença de Canavan/enzimologia , Fenótipo , Adolescente , Alelos , Amidoidrolases/química , Criança , Pré-Escolar , Análise Mutacional de DNA , Ativação Enzimática , Genótipo , Humanos , Lactente , Masculino , Modelos Moleculares , Mutação , Conformação ProteicaRESUMO
The key regulatory point of L-methionine (Met) and L-homocysteine (Hcy) degradation is catalyzed by cystathionine beta-synthase (CBS). CBS deficiency is caused by mutations in CBS gene, often resulting in protein misfolding. The prevalence of CBS deficiency in Qatar is 1/1800, â¼200-fold higher than the worldwide prevalence of 1/344 000. Almost all patients bear the CBS p.R336C variant. More than 20 years ago, it was shown in vitro that two unrelated protein variants with a substitution of an arginine (Arg) residue by cysteine (Cys) could be rescued by cysteamine (mercaptoethylamine), likely via formation of a disulfide between Cys and cysteamine, functionally mimicking the wild-type (WT) Arg side-chain. Based on these findings, we aimed to study whether cysteamine was able to improve the function of p.R336C CBS variant. Additionally, we tested the effect of mercaptoethylguanidine (MEG), a compound with a guanidino and a thiol function that may resemble Arg structure better than cysteamine. Three purified recombinant CBS proteins (p.R336C, p.R336H and WT) were pre-incubated with cysteamine, MEG or Cys (as negative control), and CBS activity and stability were measured. Pre-incubation with cysteamine and MEG increased the enzymatic activity of the p.R336C protein, which was absent upon pre-incubation with Cys. The WT and the p.R336H variant enzyme activity presented no increase with any of the tested compounds. Our results show that cysteamine and MEG are able to specifically improve the function of the CBS p.R336C variant, suggesting that any Arg-to-Cys substitution accessible to these small molecules may be converted back to a moiety resembling Arg.
Assuntos
Cistationina beta-Sintase/química , Cistationina beta-Sintase/metabolismo , Arginina/genética , Arginina/metabolismo , Western Blotting , Cistationina beta-Sintase/genética , Cisteína/genética , Cisteína/metabolismo , Fluorometria , Humanos , Estrutura Secundária de ProteínaAssuntos
Doenças do Prematuro/genética , Leucina-tRNA Ligase/genética , Falência Hepática Aguda/genética , Anormalidades Múltiplas/genética , Asfixia Neonatal/genética , Colestase/etiologia , Evolução Fatal , Retardo do Crescimento Fetal/etiologia , Humanos , Lactente , Recém-Nascido Prematuro , Doenças do Prematuro/fisiopatologia , Recém-Nascido Pequeno para a Idade Gestacional , Intestinos/anormalidades , Falência Hepática Aguda/fisiopatologia , Masculino , SíndromeRESUMO
The hexa-coordinate heme in the H2S-generating human enzyme cystathionine ß-synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO(â¢) or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO(â¢) was reported to bind at the CBS heme with very low affinity (Kd = 30-281 µm). This discrepancy was herein reconciled by investigating the NO(â¢) reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO(â¢) binds tightly to the ferrous CBS heme, with an apparent Kd ≤ 0.23 µm. In line with this result, at 25 °C, NO(â¢) binds quickly to CBS (k on â¼ 8 × 10(3) m(-1) s(-1)) and dissociates slowly from the enzyme (k off â¼ 0.003 s(-1)). The observed rate constants for NO(â¢) binding were found to be linearly dependent on [NO(â¢)] up to â¼ 800 µm NO(â¢), and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO(â¢) quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO(â¢). The novel findings reported here shed new light on CBS regulation by NO(â¢) and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO(â¢), CO, and H2S in cell signaling.