RESUMO
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that complete the first step of protein translation: ligation of amino acids to cognate tRNAs. Genes encoding ARSs have been implicated in myriad dominant and recessive phenotypes, the latter often affecting multiple tissues but with frequent involvement of the central and peripheral nervous systems, liver, and lungs. Threonyl-tRNA synthetase (TARS1) encodes the enzyme that ligates threonine to tRNATHR in the cytoplasm. To date, TARS1 variants have been implicated in a recessive brittle hair phenotype. To better understand TARS1-related recessive phenotypes, we engineered three TARS1 missense variants at conserved residues and studied these variants in Saccharomyces cerevisiae and Caenorhabditis elegans models. This revealed two loss-of-function variants, including one hypomorphic allele (R433H). We next used R433H to study the effects of partial loss of TARS1 function in a compound heterozygous mouse model (R432H/null). This model presents with phenotypes reminiscent of patients with TARS1 variants and with distinct lung and skin defects. This study expands the potential clinical heterogeneity of TARS1-related recessive disease, which should guide future clinical and genetic evaluations of patient populations.
Assuntos
Caenorhabditis elegans , Saccharomyces cerevisiae , Treonina-tRNA Ligase , Animais , Camundongos , Caenorhabditis elegans/genética , Saccharomyces cerevisiae/genética , Treonina-tRNA Ligase/genética , Treonina-tRNA Ligase/metabolismo , Humanos , Fenótipo , Mutação com Perda de Função , Modelos Animais de Doenças , Mutação de Sentido IncorretoRESUMO
The skeletal muscle is well known for its remarkable ability to regenerate after injuries. The regeneration is a complex and dynamic process that involves muscle stem cells (also called muscle satellite cells, MuSCs), fibro-adipogenic progenitors (FAPs), immune cells, and other muscle-resident cell populations. The MuSCs are the myogenic cell populaiton that contribute nuclei directly to the regenerated myofibers, while the other cell types collaboratively establish a microenvironment that facilitates myogenesis of MuSCs. The myogenic process includes activation, proliferation and differentiationof MuSCs, and subsequent fusion their descendent mononuclear myocytes into multinuclear myotubes. While the contributions of FAPs and immune cells to this microenvironment have been well studied, the influence of MuSCs on other cell types remains poorly understood. This review explores recent evidence supporting the potential role of MuSCs as immunomodulators during muscle regeneration, either through cytokine production or ligand-receptor interactions.
Assuntos
Músculo Esquelético , Regeneração , Regeneração/fisiologia , Animais , Humanos , Músculo Esquelético/fisiologia , Músculo Esquelético/citologia , Desenvolvimento Muscular , Células-Tronco/citologia , Células-Tronco/metabolismo , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/fisiologia , Diferenciação Celular , Fatores Imunológicos/farmacologia , Fatores Imunológicos/metabolismo , ImunomodulaçãoRESUMO
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that complete the first step of protein translation: ligation of amino acids to cognate tRNAs. Genes encoding ARSs have been implicated in myriad dominant and recessive phenotypes, the latter often affecting multiple tissues but with frequent involvement of the central and peripheral nervous system, liver, and lungs. Threonyl-tRNA synthetase (TARS1) encodes the enzyme that ligates threonine to tRNATHR in the cytoplasm. To date, TARS1 variants have been implicated in a recessive brittle hair phenotype. To better understand TARS1-related recessive phenotypes, we engineered three TARS1 missense mutations predicted to cause a loss-of-function effect and studied these variants in yeast and worm models. This revealed two loss-of-function mutations, including one hypomorphic allele (R433H). We next used R433H to study the effects of partial loss of TARS1 function in a compound heterozygous mouse model (R433H/null). This model presents with phenotypes reminiscent of patients with TARS1 variants and with distinct lung and skin defects. This study expands the potential clinical heterogeneity of TARS1-related recessive disease, which should guide future clinical and genetic evaluations of patient populations.
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Development is regulated by various factors, including protein methylation status. While PRMT5 is well known for its roles in oncogenesis by mediating symmetric di-methylation of arginine, its role in normal development remains elusive. Using Myod1Cre to drive Prmt5 knockout in embryonic myoblasts (Prmt5MKO), we dissected the role of PRMT5 in myogenesis. The Prmt5MKO mice are born normally but exhibit progressive muscle atrophy and premature death. Prmt5MKO inhibits proliferation and promotes premature differentiation of embryonic myoblasts, reducing the number and regenerative function of satellite cells in postnatal mice. Mechanistically, PRMT5 methylates and destabilizes FoxO1. Prmt5MKO increases the total FoxO1 level and promotes its cytoplasmic accumulation, leading to activation of autophagy and depletion of lipid droplets (LDs). Systemic inhibition of autophagy in Prmt5MKO mice restores LDs in myoblasts and moderately improves muscle regeneration. Together, PRMT5 is essential for muscle development and regeneration at least partially through mediating FoxO1 methylation and LD turnover.
Assuntos
Mioblastos , Proteína-Arginina N-Metiltransferases , Animais , Camundongos , Autofagia , Diferenciação Celular , Metilação , Mioblastos/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismoRESUMO
Transcription factors (TFs) play key roles in regulating differentiation and function of stem cells, including muscle satellite cells (MuSCs), a resident stem cell population responsible for postnatal regeneration of the skeletal muscle. Sox11 belongs to the Sry-related HMG-box (SOX) family of TFs that play diverse roles in stem cell behavior and tissue specification. Analysis of single-cell RNA-sequencing (scRNA-seq) datasets identify a specific enrichment of Sox11 mRNA in differentiating but not quiescent MuSCs. Consistent with the scRNA-seq data, Sox11 levels increase during differentiation of murine primary myoblasts in vitro. scRNA-seq data comparing muscle regeneration in young and old mice further demonstrate that Sox11 expression is reduced in aged MuSCs. Age-related decline of Sox11 expression is associated with reduced chromatin contacts within the topologically associating domains. Unexpectedly, Myod1Cre-driven deletion of Sox11 in embryonic myoblasts has no effects on muscle development and growth, resulting in apparently healthy muscles that regenerate normally. Pax7CreER- or Rosa26CreER- driven (MuSC-specific or global) deletion of Sox11 in adult mice similarly has no effects on MuSC differentiation or muscle regeneration. These results identify Sox11 as a novel myogenic differentiation marker with reduced expression in quiescent and aged MuSCs, but the specific function of Sox11 in myogenesis remains to be elucidated.
Assuntos
Músculo Esquelético , Células-Tronco , Animais , Camundongos , Diferenciação Celular , Desenvolvimento Muscular , Regeneração , Fatores de Transcrição SOXC/genéticaRESUMO
Skeletal muscle plays a key role in systemic energy homeostasis besides its contractile function, but what links these functions is poorly defined. Protein Arginine Methyl Transferase 5 (PRMT5) is a well-known oncoprotein but also expressed in healthy tissues with unclear physiological functions. As adult muscles express high levels of Prmt5, we generated skeletal muscle-specific Prmt5 knockout (Prmt5MKO ) mice. We observe reduced muscle mass, oxidative capacity, force production, and exercise performance in Prmt5MKO mice. The motor deficiency is associated with scarce lipid droplets in myofibers due to defects in lipid biosynthesis and accelerated degradation. Specifically, PRMT5 deletion reduces dimethylation and stability of Sterol Regulatory Element-Binding Transcription Factor 1a (SREBP1a), a master regulator of de novo lipogenesis. Moreover, Prmt5MKO impairs the repressive H4R3 symmetric dimethylation at the Pnpla2 promoter, elevating the level of its encoded protein ATGL, the rate-limiting enzyme catalyzing lipolysis. Accordingly, skeletal muscle-specific double knockout of Pnpla2 and Prmt5 normalizes muscle mass and function. Together, our findings delineate a physiological function of PRMT5 in linking lipid metabolism to contractile function of myofibers.
Assuntos
Proteína-Arginina N-Metiltransferases , Transferases , Animais , Camundongos , Arginina/metabolismo , Metabolismo dos Lipídeos/genética , Músculo Esquelético/metabolismo , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Transferases/metabolismoRESUMO
Transcription factors (TFs) play key roles in regulating the differentiation and function of stem cells, including muscle satellite cells (MuSCs), a resident stem cell population responsible for postnatal regeneration of the skeletal muscle. Sox11 belongs to the Sry-related HMG-box (SOX) family of TFs that play diverse roles in stem cell behavior and tissue specification. Analysis of single-cell RNA-sequencing (scRNA-seq) datasets identify a specific enrichment of Sox11 mRNA in differentiating but not quiescent MuSCs. Consistent with the scRNA-seq data, Sox11 levels increase during differentiation of murine primary myoblasts in vitro. scRNA-seq data comparing muscle regeneration in young and old mice further demonstrate that Sox11 expression is reduced in aged MuSCs. Age-related decline of Sox11 expression is associated with reduced chromatin contacts within the topologically associated domains. Unexpectedly, Myod1 Cre -driven deletion of Sox11 in embryonic myoblasts has no effects on muscle development and growth, resulting in apparently healthy muscles that regenerate normally. Pax7 CreER or Rosa26 CreER driven (MuSC-specific or global) deletion of Sox11 in adult mice similarly has no effects on MuSC differentiation or muscle regeneration. These results identify Sox11 as a novel myogenic differentiation marker with reduced expression in quiescent and aged MuSCs, but the specific function of Sox11 in myogenesis remain to be elucidated.
RESUMO
Human hematopoietic stem cells (HSCs), which arise from aorta-gonad-mesonephros (AGM), are widely used to treat blood diseases and cancers. However, a technique for their robust generation in vitro is still missing. Here we show temporal manipulation of Wnt signaling is sufficient and essential to induce AGM-like hematopoiesis from human pluripotent stem cells. TGFß inhibition at the stage of aorta-like SOX17+CD235a- hemogenic endothelium yielded AGM-like hematopoietic progenitors, which closely resembled primary cord blood HSCs at the transcriptional level and contained diverse lineage-primed progenitor populations via single cell RNA-sequencing analysis. Notably, the resulting definitive cells presented lymphoid and myeloid potential in vitro; and could home to a definitive hematopoietic site in zebrafish and rescue bloodless zebrafish after transplantation. Engraftment and multilineage repopulating activities were also observed in mouse recipients. Together, our work provided a chemically-defined and feeder-free culture platform for scalable generation of AGM-like hematopoietic progenitor cells, leading to enhanced production of functional blood and immune cells for various therapeutic applications.
Assuntos
Hemangioblastos , Animais , Diferenciação Celular/fisiologia , Hematopoese/fisiologia , Células-Tronco Hematopoéticas , Humanos , Mesonefro , Camundongos , Peixe-ZebraRESUMO
Propionate is a gut microbial metabolite that has been reported to have controversial effects on metabolic health. Here we show that propionate is activated by acyl-CoA synthetase short-chain family member 3 (ACSS3), located on the mitochondrial inner membrane in brown adipocytes. Knockout of Acss3 gene (Acss3-/- ) in mice reduces brown adipose tissue (BAT) mass but increases white adipose tissue (WAT) mass, leading to glucose intolerance and insulin resistance that are exacerbated by high-fat diet (HFD). Intriguingly, Acss3-/- or HFD feeding significantly elevates propionate levels in BAT and serum, and propionate supplementation induces autophagy in cultured brown and white adipocytes. The elevated levels of propionate in Acss3-/- mice similarly drive adipocyte autophagy, and pharmacological inhibition of autophagy using hydroxychloroquine ameliorates obesity, hepatic steatosis and insulin resistance of the Acss3-/- mice. These results establish ACSS3 as the key enzyme for propionate metabolism and demonstrate that accumulation of propionate promotes obesity and Type 2 diabetes through triggering adipocyte autophagy.
Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Coenzima A Ligases/efeitos adversos , Adipócitos Marrons/efeitos dos fármacos , Adipócitos Marrons/metabolismo , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/metabolismo , Tecido Adiposo Marrom/crescimento & desenvolvimento , Animais , Coenzima A Ligases/farmacologia , Modelos Animais de Doenças , Camundongos , Camundongos Knockout/metabolismo , Propionatos/metabolismo , Propionatos/farmacologiaRESUMO
The lipid droplet (LD) is a central hub for fatty acid metabolism in cells. Here we define the dynamics and explore the role of LDs in skeletal muscle satellite cells (SCs), a stem cell population responsible for muscle regeneration. In newly divided SCs, LDs are unequally distributed in sister cells exhibiting asymmetric cell fates, as the LDLow cell self-renews while the LDHigh cell commits to differentiation. When transplanted into regenerating muscles, LDLow cells outperform LDHigh cells in self-renewal and regeneration in vivo. Pharmacological inhibition of LD biogenesis or genetic inhibition of LD catabolism through knockout of Pnpla2 (encoding ATGL, the rate-limiting enzyme for lipolysis) disrupts cell fate homeostasis and impairs the regenerative capacity of SCs. Dysfunction of Pnpla2-null SCs is associated with energy insufficiency and oxidative stress that can be partially rescued by antioxidant (N-acetylcysteine) treatment. These results establish a direct link between LD dynamics and stem cell fate determination.
Assuntos
Gotículas Lipídicas/metabolismo , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/fisiologia , Células Satélites de Músculo Esquelético/metabolismo , Animais , Camundongos , Regeneração/fisiologiaRESUMO
Obesity and metabolic disorders caused by energy surplus pose an increasing concern within the global population. Brown adipose tissue (BAT) dissipates energy through mitochondrial non-shivering thermogenesis, thus representing a powerful agent against obesity. Here we explore the novel role of a mitochondrial outer membrane protein, LETM1-domain containing 1 (LETMD1), in BAT. We generated a knockout (Letmd1KO ) mouse model and analyzed BAT morphology, function and gene expression under various physiological conditions. While the Letmd1KO mice are born normally and have normal morphology and body weight, they lose multilocular brown adipocytes completely and have diminished mitochondrial abundance, DNA copy number, cristae structure, and thermogenic gene expression in the intrascapular BAT, associated with elevated reactive oxidative stress. In consequence, the Letmd1KO mice fail to maintain body temperature in response to acute cold exposure without food and become hypothermic within 4 h. Although the cold-exposed Letmd1KO mice can maintain body temperature in the presence of food, they cannot upregulate expression of uncoupling protein 1 (UCP1) and convert white to beige adipocytes, nor can they respond to adrenergic stimulation. These results demonstrate that LETMD1 is essential for mitochondrial structure and function, and thermogenesis of brown adipocytes.
Assuntos
Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/metabolismo , Mitocôndrias/metabolismo , Proteínas Oncogênicas/fisiologia , Receptores de Superfície Celular/fisiologia , Termogênese , Adipócitos Marrons/citologia , Tecido Adiposo Marrom/citologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismoRESUMO
Duchenne muscular dystrophy (DMD) is caused by a mutation of the muscle membrane protein dystrophin and characterized by severe degeneration of myofibers, progressive muscle wasting, loss of mobility, and, ultimately, cardiorespiratory failure and premature death. Currently there is no cure for DMD. Herein, we report that skeletal muscle-specific knockout (KO) of the phosphatase and tensin homolog (Pten) gene in an animal model of DMD (mdx mice) alleviates myofiber degeneration and restores muscle function without increasing tumor incidence. Specifically, Pten KO normalizes myofiber size and prevents muscular atrophy, and it improves grip strength and exercise performance in mdx mice. Pten KO also reduces fibrosis and inflammation, and it ameliorates muscle pathology in mdx mice. Unbiased RNA sequencing reveals that Pten KO upregulates extracellular matrix and basement membrane components positively correlated with wound healing and suppresses negative regulators of wound healing and lipid biosynthesis, thus improving the integrity of muscle basement membrane at the ultrastructural level. Importantly, pharmacological inhibition of PTEN similarly ameliorates muscle pathology and improves muscle integrity and function in mdx mice. Our findings provide evidence that PTEN inhibition may represent a potential therapeutic strategy to restore muscle function in DMD.
Assuntos
Técnicas de Silenciamento de Genes , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/genética , PTEN Fosfo-Hidrolase/genética , Regeneração/genética , Animais , Biomarcadores , Modelos Animais de Doenças , Metabolismo dos Lipídeos , Camundongos , Camundongos Endogâmicos mdx , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Músculo Esquelético/ultraestrutura , Distrofia Muscular de Duchenne/fisiopatologiaRESUMO
Single-cell RNA sequencing (scRNA-seq) is a powerful technique for deconvoluting and clustering thousands of otherwise intermingled cells based on their gene expression. Here, we present a complete protocol for the unbiased evaluation of regenerating murine skeletal muscle using scRNA-seq. The skeletal muscle is unique in its cellular composition as being primarily multinucleated muscle cells (myofibers). This protocol focuses on isolating mononuclear cells from muscle for subsequent scRNA-seq analysis and can be modified to assess cell populations in other tissues of interest. For complete details on the use and execution of this protocol, please refer to Liu et al. (2015) and Oprescu et al. (2020).
Assuntos
Músculos , RNA-Seq/métodos , Regeneração/genética , Análise de Célula Única/métodos , Animais , Células Cultivadas , Camundongos , Músculos/química , Músculos/citologia , Músculos/metabolismoRESUMO
Mammalian skeletal muscle possesses a unique ability to regenerate, which is primarily mediated by a population of resident muscle stem cells (MuSCs) and requires a concerted response from other supporting cell populations. Previous targeted analysis has described the involvement of various specific populations in regeneration, but an unbiased and simultaneous evaluation of all cell populations has been limited. Therefore, we used single-cell RNA-sequencing to uncover gene expression signatures of over 53,000 individual cells during skeletal muscle regeneration. Cells clustered into 25 populations and subpopulations, including a subpopulation of immune gene enriched myoblasts (immunomyoblasts) and subpopulations of fibro-adipogenic progenitors. Our analyses also uncovered striking spatiotemporal dynamics in gene expression, population composition, and cell-cell interaction during muscle regeneration. These findings provide insights into the cellular and molecular underpinning of skeletal muscle regeneration.
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Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly inherited diseases - where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function allele - is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapy for Charcot-Marie-Tooth disease type 2D (CMT2D), caused by dominant mutations in glycyl-tRNA synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks of age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least 1 year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof of concept for gene therapy approaches for dominant neuromuscular diseases.
Assuntos
Doença de Charcot-Marie-Tooth/terapia , Terapia Genética , Glicina-tRNA Ligase/genética , Interferência de RNA , Alelos , Animais , Modelos Animais de Doenças , Células HEK293 , Humanos , Camundongos , MutaçãoRESUMO
OBJECTIVE: To expand the clinical spectrum of lysyl-tRNA synthetase (KARS) gene-related diseases, which so far includes Charcot-Marie-Tooth disease, congenital visual impairment and microcephaly, and nonsyndromic hearing impairment. METHODS: Whole-exome sequencing was performed on index patients from 4 unrelated families with leukoencephalopathy. Candidate pathogenic variants and their cosegregation were confirmed by Sanger sequencing. Effects of mutations on KARS protein function were examined by aminoacylation assays and yeast complementation assays. RESULTS: Common clinical features of the patients in this study included impaired cognitive ability, seizure, hypotonia, ataxia, and abnormal brain imaging, suggesting that the CNS involvement is the main clinical presentation. Six previously unreported and 1 known KARS mutations were identified and cosegregated in these families. Two patients are compound heterozygous for missense mutations, 1 patient is homozygous for a missense mutation, and 1 patient harbored an insertion mutation and a missense mutation. Functional and structural analyses revealed that these mutations impair aminoacylation activity of lysyl-tRNA synthetase, indicating that defective KARS function is responsible for the phenotypes in these individuals. CONCLUSIONS: Our results demonstrate that patients with loss-of-function KARS mutations can manifest CNS disorders, thus broadening the phenotypic spectrum associated with KARS-related disease.
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Aminoacyl tRNA synthetases (ARSs) link specific amino acids with their cognate transfer RNAs in a critical early step of protein translation. Mutations in ARSs have emerged as a cause of recessive, often complex neurological disease traits. Here we report an allelic series consisting of seven novel and two previously reported biallelic variants in valyl-tRNA synthetase (VARS) in ten patients with a developmental encephalopathy with microcephaly, often associated with early-onset epilepsy. In silico, in vitro, and yeast complementation assays demonstrate that the underlying pathomechanism of these mutations is most likely a loss of protein function. Zebrafish modeling accurately recapitulated some of the key neurological disease traits. These results provide both genetic and biological insights into neurodevelopmental disease and pave the way for further in-depth research on ARS related recessive disorders and precision therapies.
Assuntos
Encefalopatias/genética , Microcefalia/genética , Valina-tRNA Ligase/genética , Alelos , Animais , Encefalopatias/enzimologia , Encefalopatias/patologia , Linhagem Celular , Modelos Animais de Doenças , Epilepsia/enzimologia , Epilepsia/genética , Epilepsia/patologia , Feminino , Fibroblastos , Técnicas de Inativação de Genes , Predisposição Genética para Doença , Humanos , Mutação com Perda de Função , Masculino , Microcefalia/enzimologia , Microcefalia/patologia , Modelos Moleculares , Transtornos do Neurodesenvolvimento/enzimologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Linhagem , Prosencéfalo/patologia , Peixe-ZebraRESUMO
This protocol details a method to analyze two tissue samples at the transcriptomic level using microarray analysis, ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA). Methods such as these provide insight into the mechanisms underlying biological differences across two samples and thus can be applied to interrogate a variety of processes across different tissue samples, conditions, and the like. The full method detailed below can be applied to determine the effects of muscle-specific Notch1 activation in the mdx mouse model and to analyze previously published microarray data of human liposarcoma cell lines.
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Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes that ligate amino acids onto tRNA molecules. Genes encoding ARSs have been implicated in phenotypically diverse dominant and recessive human diseases. The charging of tRNAPHE with phenylalanine is performed by a tetrameric enzyme that contains two alpha (FARSA) and two beta (FARSB) subunits. To date, mutations in the genes encoding these subunits (FARSA and FARSB) have not been implicated in any human disease. Here, we describe a patient with a severe, lethal, multisystem, developmental phenotype who was compound heterozygous for FARSB variants: p.Thr256Met and p.His496Lysfs*14. Expression studies using fibroblasts isolated from the proband revealed a severe depletion of both FARSB and FARSA protein levels. These data indicate that the FARSB variants destabilize total phenylalanyl-tRNA synthetase levels, thus causing a loss-of-function effect. Importantly, our patient shows strong phenotypic overlap with patients that have recessive diseases associated with other ARS loci; these observations strongly support the pathogenicity of the identified FARSB variants and are consistent with the essential function of phenylalanyl-tRNA synthetase in human cells. In sum, our clinical, genetic, and functional analyses revealed the first FARSB variants associated with a human disease phenotype and expand the locus heterogeneity of ARS-related human disease.
Assuntos
Aminoacil-tRNA Sintetases/genética , Predisposição Genética para Doença , Mutação com Perda de Função/genética , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/deficiência , Regulação da Expressão Gênica , Humanos , Masculino , Fenótipo , Fenilalanina-tRNA Ligase/genéticaRESUMO
Histidyl-tRNA synthetase (HARS) ligates histidine to cognate tRNA molecules, which is required for protein translation. Mutations in HARS cause the dominant axonal peripheral neuropathy Charcot-Marie-Tooth disease type 2W (CMT2W); however, the precise molecular mechanism remains undefined. Here, we investigated three HARS missense mutations associated with CMT2W (p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly). The three mutations localize to the HARS catalytic domain and failed to complement deletion of the yeast ortholog (HTS1). Enzyme kinetics, differential scanning fluorimetry (DSF), and analytical ultracentrifugation (AUC) were employed to assess the effect of these substitutions on primary aminoacylation function and overall dimeric structure. Notably, the p.Tyr330Cys, p.Ser356Asn, and p.Val155Gly HARS substitutions all led to reduced aminoacylation, providing a direct connection between CMT2W-linked HARS mutations and loss of canonical ARS function. While DSF assays revealed that only one of the variants (p.Val155Gly) was less thermally stable relative to wild-type, all three HARS mutants formed stable dimers, as measured by AUC. Our work represents the first biochemical analysis of CMT-associated HARS mutations and underscores how loss of the primary aminoacylation function can contribute to disease pathology.