RESUMO
WDR62 is a microcephaly-related, microtubule (MT)-associated protein (MAP) that localizes to the spindle pole and regulates spindle organization, but the underlying mechanisms remain elusive. Here, we show that WDR62 regulates spindle dynamics by recruiting katanin to the spindle pole and further reveal a TPX2-Aurora A-WDR62-katanin axis in cells. By combining cellular and in vitro experiments, we demonstrate that WDR62 shows preference for curved segments of dynamic GDP-MTs, as well as GMPCPP- and paclitaxel-stabilized MTs, suggesting that it recognizes extended MT lattice. Consistent with this property, WDR62 alone is inefficient in recruiting katanin to GDP-MTs, while WDR62 complexed with TPX2/Aurora A can potently promote katanin-mediated severing of GDP-MTs in vitro. In addition, the MT-binding affinity of WDR62 is autoinhibited through JNK phosphorylation-induced intramolecular interaction. We propose that WDR62 is an atypical MAP and functions as an adaptor protein between its recruiting factor TPX2/Aurora A and the effector katanin to orchestrate the regulation of spindle dynamics.
Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Katanina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fuso Acromático/enzimologia , Aurora Quinase A/genética , Proteínas de Ciclo Celular/genética , Células HEK293 , Células HeLa , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Katanina/genética , Cinética , Microcefalia/enzimologia , Microcefalia/genética , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Paclitaxel/farmacologia , Fosforilação , Ligação Proteica , Transporte Proteico , Transdução de Sinais , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/genética , Moduladores de Tubulina/farmacologiaRESUMO
Posttranslational modifications enhance the functional diversity of the proteome by modifying the substrates. The UFM1 cascade is a novel ubiquitin-like modification system. The mutations in UFM1, its E1 (UBA5) and E2 (UFC1), have been identified in patients with microcephaly. However, its pathological mechanisms remain unclear. Herein, we observed the disruption of the UFM1 cascade in Drosophila neuroblasts (NBs) decreased the number of NBs, leading to a smaller brain size. The lack of ufmylation in NBs resulted in an increased mitotic index and an extended G2/M phase, indicating a defect in mitotic progression. In addition, live imaging of the embryos revealed an impaired E3 ligase (Ufl1) function resulted in premature entry into mitosis and failed cellularization. Even worse, the embryonic lethality occurred as early as within the first few mitotic cycles following the depletion of Ufm1. Knockdown of ufmylation in the fixed embryos exhibited severe phenotypes, including detached centrosomes, defective microtubules, and DNA bridge. Furthermore, we observed that the UFM1 cascade could alter the level of phosphorylation on tyrosine-15 of CDK1 (pY15-CDK1), which is a negative regulator of the G2 to M transition. These findings yield unambiguous evidence suggesting that the UFM1 cascade is a microcephaly-causing factor that regulates the progression of the cell cycle at mitosis phase entry.
Assuntos
Divisão Celular , Proteínas de Drosophila , Embrião não Mamífero/enzimologia , Fase G2 , Microcefalia , Ubiquitina-Proteína Ligases , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Microcefalia/enzimologia , Microcefalia/genética , Transdução de Sinais/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Seckel syndrome (SS) is a rare spectrum of congenital severe microcephaly and dwarfism. One SS-causative gene is Ataxia Telangiectasia and Rad3-Related Protein (ATR), and ATR (c.2101 A>G) mutation causes skipping of exon 9, resulting in a hypomorphic ATR defect. This mutation is considered the cause of an impaired response to DNA replication stress, the main function of ATR, contributing to the pathogenesis of microcephaly. However, the precise behavior and impact of this splicing defect in human neural progenitor cells (NPCs) is unclear. To address this, we established induced pluripotent stem cells (iPSCs) from fibroblasts carrying the ATR mutation and an isogenic ATR-corrected counterpart iPSC clone. SS-patient-derived iPSCs (SS-iPSCs) exhibited cell type-specific splicing; exon 9 was dominantly skipped in fibroblasts and iPSC-derived NPCs, but it was included in undifferentiated iPSCs and definitive endodermal cells. SS-iPSC-derived NPCs (SS-NPCs) showed distinct expression profiles from ATR non-mutated NPCs with negative enrichment of neuronal genesis-related gene sets. In SS-NPCs, abnormal mitotic spindles occurred more frequently than in gene-corrected counterparts, and the alignment of NPCs in the surface of the neurospheres was perturbed. Finally, we tested several splicing-modifying compounds and found that TG003, a CLK1 inhibitor, could pharmacologically rescue the exon 9 skipping in SS-NPCs. Treatment with TG003 restored the ATR kinase activity in SS-NPCs and decreased the frequency of abnormal mitotic events. In conclusion, our iPSC model revealed a novel effect of the ATR mutation in mitotic processes of NPCs and NPC-specific missplicing, accompanied by the recovery of neuronal defects using a splicing rectifier.
Assuntos
Processamento Alternativo , Proteínas Mutadas de Ataxia Telangiectasia , Nanismo , Fácies , Células-Tronco Pluripotentes Induzidas , Microcefalia , Modelos Biológicos , Mutação , Proteínas Mutadas de Ataxia Telangiectasia/biossíntese , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular , Nanismo/enzimologia , Nanismo/genética , Nanismo/patologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Microcefalia/enzimologia , Microcefalia/genética , Microcefalia/patologiaRESUMO
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 (ARSs) function to transfer amino acids to cognate tRNA molecules, which are required for protein translation. To date, biallelic mutations in 31 ARS genes are known to cause recessive, early-onset severe multi-organ diseases. VARS encodes the only known valine cytoplasmic-localized aminoacyl-tRNA synthetase. Here, we report seven patients from five unrelated families with five different biallelic missense variants in VARS. Subjects present with a range of global developmental delay, epileptic encephalopathy and primary or progressive microcephaly. Longitudinal assessment demonstrates progressive cortical atrophy and white matter volume loss. Variants map to the VARS tRNA binding domain and adjacent to the anticodon domain, and disrupt highly conserved residues. Patient primary cells show intact VARS protein but reduced enzymatic activity, suggesting partial loss of function. The implication of VARS in pediatric neurodegeneration broadens the spectrum of human diseases due to mutations in tRNA synthetase genes.
Assuntos
Epilepsia/genética , Mutação , Valina-tRNA Ligase/genética , Alelos , Anticódon , Criança , Pré-Escolar , Progressão da Doença , Epilepsia/enzimologia , Epilepsia/patologia , Feminino , Predisposição Genética para Doença , Humanos , Estudos Longitudinais , Mutação com Perda de Função , Masculino , Microcefalia/enzimologia , Microcefalia/genética , Modelos Moleculares , Transtornos do Neurodesenvolvimento/enzimologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Linhagem , Biossíntese de Proteínas , Domínios e Motivos de Interação entre Proteínas , RNA de Transferência/genética , Sequenciamento do Exoma , Sequenciamento Completo do GenomaRESUMO
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
The role of protein phosphatase 2ACα (PP2ACα) in brain development is poorly understood. To understand the function of PP2ACα in neurogenesis, we inactivated Pp2acα gene in the central nervous system (CNS) of mice by Cre/LoxP system and generated the PP2ACα deficient mice (designated as the Pp2acα-/- mice). PP2ACα deletion results in DNA damage in neuroprogenitor cells (NPCs), which impairs memory formation and cortical neurogenesis. We first identify that PP2ACα can directly associate with Ataxia telangiectasia mutant kinase (ATM) and Ataxia telangiectasia/Rad3-related kinase (ATR) in neocortex and NPCs. Importantly, the P53 and hypermethylated in cancer 1 (HIC1) function complex, the newly found down-stream executor of the ATR/ATM cascade, will be translocated into nuclei and interact with homeodomain interacting protein kinase 2 (HIPK2) to respond to DNA damage. Notably, HICI plays a direct transcriptional regulatory role in HIPK2 gene expression. The interplay among P53, HIC1 and HIPK2 maintains DNA stability in neuroprogenitor cells. Taken together, our findings highlight a new role of PP2ACα in regulating early neurogenesis through maintaining DNA stability in neuroprogenitor cells. The P53/HIC/HIPK2 regulation loop, directly targeted by the ATR/ATM cascade, is involved in DNA repair in neuroprogenitor cells.
Assuntos
Encéfalo/crescimento & desenvolvimento , Dano ao DNA , Deleção de Genes , Células-Tronco Neurais/metabolismo , Proteína Fosfatase 2C/deficiência , Proteína Fosfatase 2C/genética , Animais , Encéfalo/citologia , Encéfalo/fisiologia , Proteínas de Transporte/metabolismo , Proliferação de Células , Cognição , Histonas/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Microcefalia/enzimologia , Microcefalia/genética , Neocórtex/metabolismo , Células-Tronco Neurais/citologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteína Supressora de Tumor p53/metabolismoAssuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Deficiências do Desenvolvimento/genética , Microcefalia/genética , Espasticidade Muscular/genética , Mutação , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/deficiência , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/patologia , Éxons , Expressão Gênica , Homozigoto , Humanos , Imageamento por Ressonância Magnética , Microcefalia/diagnóstico por imagem , Microcefalia/enzimologia , Microcefalia/patologia , Espasticidade Muscular/diagnóstico por imagem , Espasticidade Muscular/enzimologia , Espasticidade Muscular/patologia , Sequenciamento do ExomaRESUMO
Polynucleotide kinase-phosphatase (PNKP) is a DNA repair factor possessing both 5'-kinase and 3'-phosphatase activities to modify ends of a DNA break prior to ligation. Recently, decreased PNKP levels were identified as the cause of severe neuropathology present in the human microcephaly with seizures (MCSZ) syndrome. Utilizing novel murine Pnkp alleles that attenuate expression and a T424GfsX48 frame-shift allele identified in MCSZ individuals, we determined how PNKP inactivation impacts neurogenesis. Mice with PNKP inactivation in neural progenitors manifest neurodevelopmental abnormalities and postnatal death. This severe phenotype involved defective base excision repair and non-homologous end-joining, pathways required for repair of both DNA single- and double-strand breaks. Although mice homozygous for the T424GfsX48 allele were lethal embryonically, attenuated PNKP levels (akin to MCSZ) showed general neurodevelopmental defects, including microcephaly, indicating a critical developmental PNKP threshold. Directed postnatal neural inactivation of PNKP affected specific subpopulations including oligodendrocytes, indicating a broad requirement for genome maintenance, both during and after neurogenesis. These data illuminate the basis for selective neural vulnerability in DNA repair deficiency disease.
Assuntos
Reparo do DNA , Mutação da Fase de Leitura , Instabilidade Genômica , Células-Tronco Neurais/enzimologia , Oligodendroglia/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Animais , Humanos , Camundongos , Camundongos Mutantes , Microcefalia/enzimologia , Microcefalia/genética , Microcefalia/patologia , Células-Tronco Neurais/patologia , Oligodendroglia/patologia , Fosfotransferases (Aceptor do Grupo Álcool)/genéticaAssuntos
Centrossomo/enzimologia , Nanismo/enzimologia , Nanismo/metabolismo , Microcefalia/enzimologia , Microcefalia/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Centrossomo/patologia , Nanismo/genética , Nanismo/patologia , Predisposição Genética para Doença , Humanos , Microcefalia/genética , Microcefalia/patologia , Quinases Relacionadas a NIMA , Fenótipo , Proteínas Serina-Treonina Quinases/genética , Transdução de SinaisRESUMO
Protein translation is an essential cellular process initiated by the association of a methionyl-tRNA with the translation initiation factor eIF2. The Met-tRNA/eIF2 complex then associates with the small ribosomal subunit, other translation factors and mRNA, which together comprise the translational initiation complex. This process is regulated by the phosphorylation status of the α subunit of eIF2 (eIF2α); phosphorylated eIF2α attenuates protein translation. Here, we report a consanguineous family with severe microcephaly, short stature, hypoplastic brainstem and cord, delayed myelination and intellectual disability in two siblings. Whole-exome sequencing identified a homozygous missense mutation, c.1972G>A; p.Arg658Cys, in protein phosphatase 1, regulatory subunit 15b (PPP1R15B), a protein which functions with the PPP1C phosphatase to maintain dephosphorylated eIF2α in unstressed cells. The p.R658C PPP1R15B mutation is located within the PPP1C binding site. We show that patient cells have greatly diminished levels of PPP1R15B-PPP1C interaction, which results in increased eIF2α phosphorylation and resistance to cellular stress. Finally, we find that patient cells have elevated levels of PPP1R15B mRNA and protein, suggesting activation of a compensatory program aimed at restoring cellular homeostasis which is ineffective due to PPP1R15B alteration. PPP1R15B now joins the expanding list of translation-associated proteins which when mutated cause rare genetic diseases.
Assuntos
Nanismo/genética , Fator de Iniciação 2 em Eucariotos/genética , Deficiência Intelectual/genética , Proteína Fosfatase 1/genética , Sítios de Ligação , Estatura/genética , Proteínas de Ciclo Celular/genética , Pré-Escolar , Consanguinidade , Nanismo/enzimologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Feminino , Homozigoto , Humanos , Deficiência Intelectual/enzimologia , Masculino , Microcefalia/enzimologia , Microcefalia/genética , Mutação , Mutação de Sentido Incorreto , Fosforilação , Biossíntese de Proteínas , Proteína Fosfatase 1/metabolismo , Subunidades Proteicas , Análise de Sequência de DNARESUMO
BACKGROUND: Heterozygous loss-of-function mutations in the X-linked CASK gene cause progressive microcephaly with pontine and cerebellar hypoplasia (MICPCH) and severe intellectual disability (ID) in females. Different CASK mutations have also been reported in males. The associated phenotypes range from nonsyndromic ID to Ohtahara syndrome with cerebellar hypoplasia. However, the phenotypic spectrum in males has not been systematically evaluated to date. METHODS: We identified a CASK alteration in 8 novel unrelated male patients by targeted Sanger sequencing, copy number analysis (MLPA and/or FISH) and array CGH. CASK transcripts were investigated by RT-PCR followed by sequencing. Immunoblotting was used to detect CASK protein in patient-derived cells. The clinical phenotype and natural history of the 8 patients and 28 CASK-mutation positive males reported previously were reviewed and correlated with available molecular data. RESULTS: CASK alterations include one nonsense mutation, one 5-bp deletion, one mutation of the start codon, and five partial gene deletions and duplications; seven were de novo, including three somatic mosaicisms, and one was familial. In three subjects, specific mRNA junction fragments indicated in tandem duplication of CASK exons disrupting the integrity of the gene. The 5-bp deletion resulted in multiple aberrant CASK mRNAs. In fibroblasts from patients with a CASK loss-of-function mutation, no CASK protein could be detected. Individuals who are mosaic for a severe CASK mutation or carry a hypomorphic mutation still showed detectable amount of protein. CONCLUSIONS: Based on eight novel patients and all CASK-mutation positive males reported previously three phenotypic groups can be distinguished that represent a clinical continuum: (i) MICPCH with severe epileptic encephalopathy caused by hemizygous loss-of-function mutations, (ii) MICPCH associated with inactivating alterations in the mosaic state or a partly penetrant mutation, and (iii) syndromic/nonsyndromic mild to severe ID with or without nystagmus caused by CASK missense and splice mutations that leave the CASK protein intact but likely alter its function or reduce the amount of normal protein. Our findings facilitate focused testing of the CASK gene and interpreting sequence variants identified by next-generation sequencing in cases with a phenotype resembling either of the three groups.
Assuntos
Guanilato Quinases/genética , Microcefalia/enzimologia , Adolescente , Adulto , Cerebelo/anormalidades , Cerebelo/enzimologia , Criança , Pré-Escolar , Deficiências do Desenvolvimento/enzimologia , Deficiências do Desenvolvimento/etiologia , Deficiências do Desenvolvimento/genética , Humanos , Lactente , Deficiência Intelectual/enzimologia , Deficiência Intelectual/etiologia , Deficiência Intelectual/genética , Masculino , Microcefalia/complicações , Microcefalia/genética , Pessoa de Meia-Idade , Mutação , Malformações do Sistema Nervoso/enzimologia , Malformações do Sistema Nervoso/etiologia , Malformações do Sistema Nervoso/genética , Fenótipo , Adulto JovemRESUMO
Protein phosphorylation is a nearly universal post-translation modification involved in a plethora of cellular events. Even though phosphorylation of extracellular proteins had been observed, the identity of the kinases that phosphorylate secreted proteins remained a mystery until only recently. Advances in genome sequencing and genetic studies have paved the way for the discovery of a new class of kinases that localize within the endoplasmic reticulum, Golgi apparatus and the extracellular space. These novel kinases phosphorylate proteins and proteoglycans in the secretory pathway and appear to regulate various extracellular processes. Mutations in these kinases cause human disease, thus underscoring the biological importance of phosphorylation within the secretory pathway. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.
Assuntos
Anormalidades Múltiplas/genética , Caseína Quinase I/genética , Fissura Palatina/genética , Exoftalmia/genética , Proteínas da Matriz Extracelular/genética , Microcefalia/genética , Osteosclerose/genética , Proteínas Tirosina Quinases/genética , Via Secretória/genética , Anormalidades Múltiplas/enzimologia , Animais , Caseína Quinase I/química , Caseína Quinase I/metabolismo , Fissura Palatina/enzimologia , Retículo Endoplasmático/enzimologia , Retículo Endoplasmático/genética , Exoftalmia/enzimologia , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Complexo de Golgi/enzimologia , Complexo de Golgi/genética , Humanos , Microcefalia/enzimologia , Mutação , Osteosclerose/enzimologia , Fosforilação/genética , Conformação Proteica , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Especificidade por SubstratoRESUMO
Several studies have recently shown that germline mutations in RTEL1, an essential DNA helicase involved in telomere regulation and DNA repair, cause Hoyeraal-Hreidarsson syndrome (HHS), a severe form of dyskeratosis congenita. Using original new softwares, facilitating the delineation of the different domains of the protein and the identification of remote relationships for orphan domains, we outline here that the C-terminal extension of RTEL1, downstream of its catalytic domain and including several HHS-associated mutations, contains a yet unidentified tandem of harmonin-N-like domains, which may serve as a hub for partner interaction. This finding highlights the potential critical role of this region for the function of RTEL1 and gives insights into the impact that the identified mutations would have on the structure and function of these domains.
Assuntos
DNA Helicases/química , Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Deficiência Intelectual/genética , Microcefalia/genética , Sequência de Aminoácidos , Sequência Conservada , DNA Helicases/genética , Disceratose Congênita/enzimologia , Retardo do Crescimento Fetal/enzimologia , Duplicação Gênica , Mutação em Linhagem Germinativa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Deficiência Intelectual/enzimologia , Microcefalia/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de ProteínaRESUMO
We analyzed four families that presented with a similar condition characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. We show that recessive mutations in the ASNS gene are responsible for this syndrome. Two of the identified missense mutations dramatically reduce ASNS protein abundance, suggesting that the mutations cause loss of function. Hypomorphic Asns mutant mice have structural brain abnormalities, including enlarged ventricles and reduced cortical thickness, and show deficits in learning and memory mimicking aspects of the patient phenotype. ASNS encodes asparagine synthetase, which catalyzes the synthesis of asparagine from glutamine and aspartate. The neurological impairment resulting from ASNS deficiency may be explained by asparagine depletion in the brain or by accumulation of aspartate/glutamate leading to enhanced excitability and neuronal damage. Our study thus indicates that asparagine synthesis is essential for the development and function of the brain but not for that of other organs.
Assuntos
Aspartato-Amônia Ligase/deficiência , Aspartato-Amônia Ligase/genética , Encéfalo/enzimologia , Encéfalo/patologia , Predisposição Genética para Doença/genética , Microcefalia/enzimologia , Microcefalia/genética , Adolescente , Animais , Atrofia/complicações , Atrofia/enzimologia , Atrofia/genética , Criança , Feminino , Humanos , Lactente , Recém-Nascido , Deficiência Intelectual/complicações , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Deficiência Intelectual/patologia , Masculino , Camundongos , Camundongos Transgênicos , Microcefalia/complicações , Microcefalia/patologia , Mutação de Sentido Incorreto/genética , Linhagem , SíndromeRESUMO
The DNA-dependent protein kinase catalytic subunit (DNA-PKcs; encoded by PRKDC) functions in DNA non-homologous end-joining (NHEJ), the major DNA double strand break (DSB) rejoining pathway. NHEJ also functions during lymphocyte development, joining V(D)J recombination intermediates during antigen receptor gene assembly. Here, we describe a patient with compound heterozygous mutations in PRKDC, low DNA-PKcs expression, barely detectable DNA-PK kinase activity, and impaired DSB repair. In a heterologous expression system, we found that one of the PRKDC mutations inactivated DNA-PKcs, while the other resulted in dramatically diminished but detectable residual function. The patient suffered SCID with reduced or absent T and B cells, as predicted from PRKDC-deficient animal models. Unexpectedly, the patient was also dysmorphic; showed severe growth failure, microcephaly, and seizures; and had profound, globally impaired neurological function. MRI scans revealed microcephaly-associated cortical and hippocampal dysplasia and progressive atrophy over 2 years of life. These neurological features were markedly more severe than those observed in patients with deficiencies in other NHEJ proteins. Although loss of DNA-PKcs in mice, dogs, and horses was previously shown not to impair neuronal development, our findings demonstrate a stringent requirement for DNA-PKcs during human neuronal development and suggest that high DNA-PK protein expression is required to sustain efficient pre- and postnatal neurogenesis.
Assuntos
Anormalidades Múltiplas/diagnóstico , Encéfalo/anormalidades , Proteína Quinase Ativada por DNA/genética , Microcefalia/diagnóstico , Proteínas Nucleares/genética , Imunodeficiência Combinada Severa/diagnóstico , Anormalidades Múltiplas/enzimologia , Anormalidades Múltiplas/genética , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular , Pré-Escolar , Sequência Conservada , Análise Mutacional de DNA , Reparo do DNA , Evolução Fatal , Estudos de Associação Genética , Humanos , Masculino , Microcefalia/enzimologia , Microcefalia/genética , Técnicas de Diagnóstico Molecular , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Mutação Puntual , Imunodeficiência Combinada Severa/enzimologia , Imunodeficiência Combinada Severa/genéticaRESUMO
BACKGROUND: Kaufman oculocerebrofacial syndrome (KOS) is a developmental disorder characterised by reduced growth, microcephaly, ocular anomalies (microcornea, strabismus, myopia, and pale optic disk), distinctive facial features (narrow palpebral fissures, telecanthus, sparse and laterally broad eyebrows, preauricular tags, and micrognathia), mental retardation, and generalised hypotonia. KOS is a rare, possibly underestimated condition, with fewer than 10 cases reported to date. Here we investigate the molecular cause underlying KOS. METHODS: An exome sequencing approach was used on a single affected individual of an Italian consanguineous family coupled with mutation scanning using Sanger sequencing on a second unrelated subject with clinical features fitting the disorder. RESULTS: Exome sequencing was able to identify homozygosity for a novel truncating mutation (c.556C>T, p.Arg186stop) in UBE3B, which encodes a widely expressed HECT (homologous to the E6-AP carboxyl terminus) domain E3 ubiquitin-protein ligase. Homozygosity for a different nonsense lesion affecting the gene (c.1166G>A, p.Trp389stop) was documented in the second affected subject, supporting the recessive mode of inheritance of the disorder. Mutation scanning of the entire UBE3B coding sequence on a selected cohort of subjects with features overlapping, in part, those recurring in KOS did not reveal disease-causing mutations, suggesting phenotypic homogeneity of UBE3B lesions. DISCUSSION: Our data provide evidence that KOS is caused by UBE3B loss of function, and further demonstrate the impact of misregulation of protein ubiquitination on development and growth. The available clinical records, including those referring to four UBE3B mutation-positive subjects recently described as belonging to a previously unreported entity, which fits KOS, document the clinical homogeneity of this disorder.
Assuntos
Anormalidades do Olho/enzimologia , Anormalidades do Olho/genética , Deficiência Intelectual/enzimologia , Deficiência Intelectual/genética , Deformidades Congênitas dos Membros/enzimologia , Deformidades Congênitas dos Membros/genética , Microcefalia/enzimologia , Microcefalia/genética , Ubiquitina-Proteína Ligases/genética , Sequência de Bases , Criança , Exoma , Fácies , Feminino , Homozigoto , Humanos , Masculino , Dados de Sequência Molecular , Mutação , LinhagemRESUMO
The mammalian target of rapamycin (mTOR) assembles into two distinct multi-protein complexes called mTORC1 and mTORC2. Whereas mTORC1 is known to regulate cell and organismal growth, the role of mTORC2 is less understood. We describe two mouse lines that are devoid of the mTORC2 component rictor in the entire central nervous system or in Purkinje cells. In both lines neurons were smaller and their morphology and function were strongly affected. The phenotypes were accompanied by loss of activation of Akt, PKC, and SGK1 without effects on mTORC1 activity. The striking decrease in the activation and expression of several PKC isoforms, the subsequent loss of activation of GAP-43 and MARCKS, and the established role of PKCs in spinocerebellar ataxia and in shaping the actin cytoskeleton strongly suggest that the morphological deficits observed in rictor-deficient neurons are mediated by PKCs. Together our experiments show that mTORC2 has a particularly important role in the brain and that it affects size, morphology, and function of neurons.
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Encéfalo/metabolismo , Proteínas de Transporte/metabolismo , Forma Celular , Tamanho Celular , Complexos Multiproteicos/metabolismo , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Western Blotting , Encéfalo/enzimologia , Encéfalo/patologia , Contagem de Células , Cerebelo/enzimologia , Cerebelo/patologia , Ativação Enzimática , Deleção de Genes , Alvo Mecanístico do Complexo 1 de Rapamicina , Alvo Mecanístico do Complexo 2 de Rapamicina , Camundongos , Camundongos Knockout , Microcefalia/enzimologia , Microcefalia/patologia , Fenótipo , Células de Purkinje/enzimologia , Proteína Companheira de mTOR Insensível à Rapamicina , Sinapses/metabolismoRESUMO
Ataxia-telangiectasia and rad3 (ATR)-related Seckel syndrome is associated with growth retardation and premature aging features. ATR-Seckel fibroblasts have a reduced replicative capacity in vitro and an aged morphology that is associated with activation of stress-associated p38 mitogen-activated protein kinase and phosphorylated HSP27. These phenotypes are prevented using p38 inhibitors, with replicative capacity restored to the normal range. However, this stressed phenotype is retained in telomerase-immortalized ATR-Seckel fibroblasts, indicating that it is independent of telomere erosion. As with normal fibroblasts, senescence in ATR-Seckel is bypassed by p53 abrogation. Young ATR-Seckel fibroblasts show elevated levels of p21(WAF1), p16(INK4A), phosphorylated actin-binding protein cofilin, and phosphorylated caveolin-1, with small molecule drug inhibition of p38 reducing p16(INK4A) and caveolin-1 phosphorylation. In conclusion, ATR-Seckel fibroblasts undergo accelerated aging via stress-induced premature senescence and p38 activation that may underlie certain clinical features of Seckel syndrome, and our data suggest a novel target for pharmacological intervention in this human syndrome.