RESUMO
Alternative splicing (AS) appears to be altered in Huntington's disease (HD), but its significance for early, pre-symptomatic disease stages has not been inspected. Here, taking advantage of Htt CAG knock-in mouse in vitro and in vivo models, we demonstrate a correlation between Htt CAG repeat length and increased aberrant linear AS, specifically affecting neural progenitors and, in vivo, the striatum prior to overt behavioral phenotypes stages. Remarkably, a significant proportion (36%) of the aberrantly spliced isoforms are not-functional and meant to non-sense mediated decay (NMD). The expanded Htt CAG repeats further reflect on a previously neglected, global impairment of back-splicing, leading to decreased circular RNAs production in neural progenitors. Integrative transcriptomic analyses unveil a network of transcriptionally altered micro-RNAs and RNA-binding proteins (Celf, hnRNPs, Ptbp, Srsf, Upf1, Ythd2) which might influence the AS machinery, primarily in neural cells. We suggest that this unbalanced expression of linear and circular RNAs might alter neural fitness, contributing to HD pathogenesis.
Assuntos
Doença de Huntington , Camundongos , Animais , Doença de Huntington/genética , Doença de Huntington/patologia , RNA Circular/genética , Splicing de RNA , Processamento Alternativo/genética , Perfilação da Expressão Gênica , Expansão das Repetições de Trinucleotídeos/genética , Proteína Huntingtina/genéticaRESUMO
Autosomal dominant mutations in the gene encoding α-synuclein (SNCA) were the first to be linked with hereditary Parkinson's disease (PD). Duplication and triplication of SNCA has been observed in PD patients, together with mutations at the N-terminal of the protein, among which A30P and A53T influence the formation of fibrils. By overexpressing human α-synuclein in the neuronal system of Drosophila, we functionally validated the ability of IP3K2, an ortholog of the GWAS identified risk gene, Inositol-trisphosphate 3-kinase B (ITPKB), to modulate α-synuclein toxicity in vivo. ITPKB mRNA and protein levels were also increased in SK-N-SH cells overexpressing wild-type α-synuclein, A53T or A30P mutants. Kinase overexpression was detected in the cytoplasmatic and in the nuclear compartments in all α-synuclein cell types. By quantifying mRNAs in the cortex of PD patients, we observed higher levels of ITPKB mRNA when SNCA was expressed more (p < 0.05), compared to controls. A positive correlation was also observed between SNCA and ITPKB expression in the cortex of patients, which was not seen in the controls. We replicated this observation in a public dataset. Our data, generated in SK-N-SH cells and in cortex from PD patients, show that the expression of α-synuclein and ITPKB is correlated in pathological situations.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Mutação , Neurônios/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismoRESUMO
Disruptive mutations in the chromodomain helicase DNA-binding protein 8 gene (CHD8) have been recurrently associated with autism spectrum disorders (ASDs). Here we investigated how chromatin reacts to CHD8 suppression by analyzing a panel of histone modifications in induced pluripotent stem cell-derived neural progenitors. CHD8 suppression led to significant reduction (47.82%) in histone H3K36me3 peaks at gene bodies, particularly impacting on transcriptional elongation chromatin states. H3K36me3 reduction specifically affects highly expressed, CHD8-bound genes and correlates with altered alternative splicing patterns of 462 genes implicated in 'regulation of RNA splicing' and 'mRNA catabolic process'. Mass spectrometry analysis uncovered a novel interaction between CHD8 and the splicing regulator heterogeneous nuclear ribonucleoprotein L (hnRNPL), providing the first mechanistic insights to explain the CHD8 suppression-derived splicing phenotype, partly implicating SETD2, a H3K36me3 methyltransferase. In summary, our results point toward broad molecular consequences of CHD8 suppression, entailing altered histone deposition/maintenance and RNA processing regulation as important regulatory processes in ASD.
Assuntos
Processamento Alternativo , Caderinas , Histonas , Cromatina , Histonas/metabolismo , Lisina/metabolismo , RNA/metabolismo , Caderinas/genética , Humanos , Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Transtorno do Espectro Autista/genéticaRESUMO
Mutations in the SZT2 gene have been associated with developmental and epileptic encephalopathy-18, a rare severe autosomal recessive neurologic disorder, characterized by psychomotor impairment/intellectual disability, dysmorphic facial features and early onset of refractory seizures. Here we report the generation of the first induced pluripotent stem cell (iPSC) lines from a patient with treatment-resistant epilepsy, carrying compound heterozygous mutations in SZT2 (Mut1: c.498G>T and Mut2: c.6553C>T), and his healthy heterozygous parents. Peripheral blood mononuclear cells were reprogrammed by a non-integrating Sendai virus-based reprogramming system. The generated human iPSC lines exhibited expression of the main pluripotency markers, the potential to differentiate into all three germ layers and presented a normal karyotype. These lines represent a valuable resource to study neurodevelopmental alterations, and to obtain mature, pathology-relevant neuronal populations as an in vitro model to perform functional assays and test the patient's responsiveness to novel antiepileptic treatments.
Assuntos
Epilepsia Generalizada , Células-Tronco Pluripotentes Induzidas , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares , Mutação , Heterozigoto , Proteínas do Tecido Nervoso/metabolismoRESUMO
SINEUP is a new class of long non-coding RNAs (lncRNAs) which contain an inverted Short Interspersed Nuclear Element (SINE) B2 element (invSINEB2) necessary to specifically upregulate target gene translation. Originally identified in the mouse AS-Uchl1 (antisense Ubiquitin carboxyl-terminal esterase L1) locus, natural SINEUP molecules are oriented head to head to their sense protein coding, target gene (Uchl1, in this example). Peculiarly, SINEUP is able to augment, in a specific and controlled way, the expression of the target protein, with no alteration of target mRNA levels. SINEUP is characterized by a modular structure with the Binding Domain (BD) providing specificity to the target transcript and an effector domain (ED)-containing the invSINEB2 element-able to promote the loading to the heavy polysomes of the target mRNA. Since the understanding of its modular structure in the endogenous AS-Uchl1 ncRNA, synthetic SINEUP molecules have been developed by creating a specific BD for the gene of interest and placing it upstream the invSINEB2 ED. Synthetic SINEUP is thus a novel molecular tool that potentially may be used for any industrial or biomedical application to enhance protein production, also as possible therapeutic strategy in haploinsufficiency-driven disorders.Here, we describe a detailed protocol to (1) design a specific BD directed to a gene of interest and (2) assemble and clone it with the ED to obtain a functional SINEUP molecule. Then, we provide guidelines to efficiently deliver SINEUP into mammalian cells and evaluate its ability to effectively upregulate target protein translation.
Assuntos
Biossíntese de Proteínas , RNA Longo não Codificante , Animais , Camundongos , RNA Antissenso/genética , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos Nucleotídeos Curtos e DispersosRESUMO
CHD8 represents one of the highest confidence genetic risk factors implied in Autism Spectrum Disorders, with most mutations leading to CHD8 haploinsufficiency and the insurgence of specific phenotypes, such as macrocephaly, facial dysmorphisms, intellectual disability, and gastrointestinal complaints. While extensive studies have been conducted on the possible consequences of CHD8 suppression and protein coding RNAs dysregulation during neuronal development, the effects of transcriptional changes of long non-coding RNAs (lncRNAs) remain unclear. In this study, we focused on a peculiar class of natural antisense lncRNAs, SINEUPs, that enhance translation of a target mRNA through the activity of two RNA domains, an embedded transposable element sequence and an antisense region. By looking at dysregulated transcripts following CHD8 knock down (KD), we first identified RAB11B-AS1 as a potential SINEUP RNA for its domain configuration. Then we demonstrated that such lncRNA is able to increase endogenous RAB11B protein amounts without affecting its transcriptional levels. RAB11B has a pivotal role in vesicular trafficking, and mutations on this gene correlate with intellectual disability and microcephaly. Thus, our study discloses an additional layer of molecular regulation which is altered by CHD8 suppression. This represents the first experimental confirmation that naturally occurring SINEUP could be involved in ASD pathogenesis and underscores the importance of dysregulation of functional lncRNAs in neurodevelopment.
RESUMO
Huntington's disease (HD) is a genetic, fatal autosomal dominant neurodegenerative disorder typically occurring in midlife with symptoms ranging from chorea, to dementia, to personality disturbances (Philos Trans R Soc Lond Ser B Biol Sci 354:957-961, 1999). HD is inherited in a dominant fashion, and the underlying mutation in all cases is a CAG trinucleotide repeat expansion within exon 1 of the HD gene (Cell 72:971-983, 1993). The expanded CAG repeat, translated into a lengthened glutamine tract at the amino terminus of the huntingtin protein, affects its structural properties and functional activities. The effects are pleiotropic, as huntingtin is broadly expressed in different cellular compartments (i.e., cytosol, nucleus, mitochondria) as well as in all cell types of the body at all developmental stages, such that HD pathogenesis likely starts at conception and is a lifelong process (Front Neurosci 9:509, 2015). The rate-limiting mechanism(s) of neurodegeneration in HD still remains elusive: many different processes are commonly disrupted in HD cell lines and animal models, as well as in HD patient cells (Eur J Neurosci 27:2803-2820, 2008); however, epigenetic-chromatin deregulation, as determined by the analysis of DNA methylation, histone modifications, and noncoding RNAs, has now become a prevailing feature. Thus, the overarching goal of this chapter is to discuss the current status of the literature, reviewing how an aberrant epigenetic landscape can contribute to altered gene expression and neuronal dysfunction in HD.
Assuntos
Epigênese Genética/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Acetilação , Animais , Linhagem Celular , Montagem e Desmontagem da Cromatina/genética , Ensaios Clínicos como Assunto , Metilação de DNA/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica/genética , Código das Histonas/genética , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/fisiologia , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/fisiologia , Homeostase , Humanos , Doença de Huntington/tratamento farmacológico , Doença de Huntington/metabolismo , Metilação , Proteína de Leucina Linfoide-Mieloide/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Proteínas do Grupo Polycomb/fisiologia , Processamento de Proteína Pós-Traducional/genética , RNA não Traduzido/genéticaRESUMO
Human SH-SY5Y neuroblastoma cells are widely utilized in in vitro studies to dissect out pathogenetic mechanisms of neurodegenerative disorders. These cells are considered as neuronal precursors and differentiate into more mature neuronal phenotypes under selected growth conditions. In this study, in order to decipher the pathways and cellular processes underlying neuroblastoma cell differentiation in vitro, we performed systematic transcriptomic (RNA-seq) and bioinformatic analysis of SH-SY5Y cells differentiated according to a two-step paradigm: retinoic acid treatment followed by enriched neurobasal medium. Categorization of 1989 differentially expressed genes (DEGs) identified in differentiated cells functionally linked them to changes in cell morphology including remodelling of plasma membrane and cytoskeleton, and neuritogenesis. Seventy-three DEGs were assigned to axonal guidance signalling pathway, and the expression of selected gene products such as neurotrophin receptors, the functionally related SLITRK6, and semaphorins, was validated by immunoblotting. Along with these findings, the differentiated cells exhibited an ability to elongate longer axonal process as assessed by the neuronal cytoskeletal markers biochemical characterization and morphometric evaluation. Recognition of molecular events occurring in differentiated SH-SY5Y cells is critical to accurately interpret the cellular responses to specific stimuli in studies on disease pathogenesis.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Neuroblastoma/metabolismo , Neurônios/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Perfilação da Expressão Gênica/métodos , Humanos , Proteínas de Membrana/metabolismo , Neuroblastoma/tratamento farmacológico , Neurônios/citologia , Neurônios/efeitos dos fármacos , Tretinoína/farmacologiaRESUMO
Pathogenic bacteria produce powerful virulent factors, such as pore-forming toxins, that promote their survival and cause serious damage to the host. Host cells reply to membrane stresses and ionic imbalance by modifying gene expression at the epigenetic, transcriptional and translational level, to recover from the toxin attack. The fact that the majority of the human transcriptome encodes for non-coding RNAs (ncRNAs) raises the question: do host cells deploy non-coding transcripts to rapidly control the most energy-consuming process in cells-i.e., host translation-to counteract the infection? Here, we discuss the intriguing possibility that membrane-damaging toxins induce, in the host, the expression of toxin-specific long non-coding RNAs (lncRNAs), which act as sponges for other molecules, encoding small peptides or binding target mRNAs to depress their translation efficiency. Unravelling the function of host-produced lncRNAs upon bacterial infection or membrane damage requires an improved understanding of host lncRNA expression patterns, their association with polysomes and their function during this stress. This field of investigation holds a unique opportunity to reveal unpredicted scenarios and novel approaches to counteract antibiotic-resistant infections.
Assuntos
Infecções Bacterianas/genética , Interações Hospedeiro-Patógeno , RNA Longo não Codificante , Fenômenos Fisiológicos Bacterianos , Toxinas Bacterianas/metabolismo , Expressão Gênica , Humanos , Biossíntese de ProteínasRESUMO
Arnica montana (Arnica m.) is used for its purported anti-inflammatory and tissue healing actions after trauma, bruises, or tissue injuries, but its cellular and molecular mechanisms are largely unknown. This work tested Arnica m. effects on gene expression using an in vitro model of macrophages polarized towards a "wound-healing" phenotype. The monocyte-macrophage human THP-1 cell line was cultured and differentiated with phorbol-myristate acetate and Interleukin-4, then exposed for 24h to Arnica m. centesimal (c) dilutions 2c, 3c, 5c, 9c, 15c or Control. Total RNA was isolated and cDNA libraries were sequenced with a NextSeq500 sequencer. Genes with significantly positive (up-regulated) or negative (down-regulated) fold changes were defined as differentially expressed genes (DEGs). A total of 20 DEGs were identified in Arnica m. 2c treated cells. Of these, 7 genes were up-regulated and 13 were down-regulated. The most significantly up-regulated function concerned 4 genes with a conserved site of epidermal growth factor-like region (p<0.001) and three genes of proteinaceous extracellular matrix, including heparin sulphate proteoglycan 2 (HSPG2), fibrillin 2 (FBN2), and fibronectin (FN1) (p<0.01). Protein assay confirmed a statistically significant increase of fibronectin production (p<0.05). The down-regulated transcripts derived from mitochondrial genes coding for some components of electron transport chain. The same groups of genes were also regulated by increasing dilutions of Arnica m. (3c, 5c, 9c, 15c), although with a lower effect size. We further tested the healing potential of Arnica m. 2c in a scratch model of wound closure based on the motility of bone marrow-derived macrophages and found evidence of an accelerating effect on cell migration in this system. The results of this work, taken together, provide new insights into the action of Arnica m. in tissue healing and repair, and identify extracellular matrix regulation by macrophages as a therapeutic target.
Assuntos
Anti-Inflamatórios/farmacologia , Arnica/química , Matriz Extracelular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Cicatrização/efeitos dos fármacos , Anti-Inflamatórios/química , Linhagem Celular , Matriz Extracelular/genética , Humanos , Macrófagos/metabolismo , Extratos Vegetais/químicaRESUMO
Fourteen mycobacterial strains isolated from pulmonary samples of independent patients in the state of Pará (Brazil), and three strains isolated in Italy, were characterized using a polyphasic approach. Thorough genetic investigation, including whole-genome sequencing, demonstrated that the strains belong to the M. simiae complex, being most closely related to Mycobacterium interjectum. For 14 of the strains, evidence emerged supporting their inclusion in a previously unreported species of the genus Mycobacterium, for which the name Mycobacterium paraense sp. nov. is proposed (type strain, IEC26(T)â= DSM 46749(T)â= CCUG 66121(T)). The novel species is characterized by slow growth, unpigmented or pale yellow scotochromogenic colonies, and a HPLC mycolic acid profile different from other known mycobacteria. In different genetic regions, high sequence microheterogeneity was detected.
Assuntos
Infecções por Mycobacterium/microbiologia , Mycobacterium/classificação , Filogenia , Técnicas de Tipagem Bacteriana , Brasil , DNA Bacteriano/genética , Genes Bacterianos , Humanos , Itália , Dados de Sequência Molecular , Mycobacterium/genética , Mycobacterium/isolamento & purificação , Ácidos Micólicos/química , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Escarro/microbiologiaRESUMO
The mechanotransduction process in hair cells in the inner ear is associated with the influx of calcium from the endolymph. Calcium is exported back to the endolymph via the splice variant w/a of the PMCA2 of the stereocilia membrane. To further investigate the role of the pump, we have identified and characterized a novel ENU-induced mouse mutation, Tommy, in the PMCA2 gene. The mutation causes a non-conservative E629K change in the second intracellular loop of the pump that harbors the active site. Tommy mice show profound hearing impairment from P18, with significant differences in hearing thresholds between wild type and heterozygotes. Expression of mutant PMCA2 in CHO cells shows calcium extrusion impairment; specifically, the long term, non-stimulated calcium extrusion activity of the pump is inhibited. Calcium extrusion was investigated directly in neonatal organotypic cultures of the utricle sensory epithelium in Tommy mice. Confocal imaging combined with flash photolysis of caged calcium showed impairment of calcium export in both Tommy heterozygotes and homozygotes. Immunofluorescence studies of the organ of Corti in homozygous Tommy mice showed a progressive base to apex degeneration of hair cells after P40. Our results on the Tommy mutation along with previously observed interactions between cadherin-23 and PMCA2 mutations in mouse and humans underline the importance of maintaining the appropriate calcium concentrations in the endolymph to control the rigidity of cadherin and ensure the function of interstereocilia links, including tip links, of the stereocilia bundle.
Assuntos
Cálcio/metabolismo , Citosol/metabolismo , Surdez/genética , Surdez/metabolismo , Células Ciliadas Auditivas/metabolismo , Mutação de Sentido Incorreto , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Sequência de Aminoácidos , Animais , Citosol/química , Modelos Animais de Doenças , Orelha Interna/metabolismo , Células Ciliadas Auditivas/química , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Alinhamento de SequênciaRESUMO
Acidic phospholipids increase the affinity of the plasma membrane Ca(2+)-ATPase pump for Ca(2+). They interact with the C-terminal region of the pump and with a domain in the loop connecting transmembrane domains 2 and 3 (A(L) region) next to site A of alternative splicing. The contribution of the two phospholipid-binding sites and the possible interference of splicing inserts at site A with the regulation of the ATPase activity of isoform 2 of the pump by phospholipids have been analyzed. The activity of the full-length z/b variant (no insert at site A), the w/b (with insert at site A), and the w/a variant, containing both the 45-amino acid A-site insert and a C-site insert that truncates the pump in the calmodulin binding domain, has been analyzed in microsomal membranes of overexpressing CHO cells. The A-site insertion did not modify the phospholipid sensitivity of the pump, but the doubly inserted w/a variant became insensitive to acidic phospholipids, even if containing the intact A(L) phospholipid binding domain. Pump mutants in which 12 amino acids had been deleted, or single lysine mutations introduced, in the A(L) region were studied by monitoring agonist-induced Ca(2+) transients in overexpressing CHO cells. The 12-residue deletion completely abolished the ATPase activity of the w/a variant but only reduced that of the z/b variant, which was also affected by the single lysine substitutions in the same domain. A structural interpretation of the interplay of the pump with phospholipids, and of the mechanism of their activation, is proposed on the basis of molecular modeling studies.
Assuntos
Processamento Alternativo , Cálcio/metabolismo , Modelos Moleculares , Fosfolipídeos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Microssomos , Mutação de Sentido Incorreto , Fosfolipídeos/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Estrutura Terciária de Proteína , Ratos , Deleção de SequênciaRESUMO
The aim of this study was to screen 349 patients affected by sensorineural hearing loss (SNHL), mostly from the Campania region (southern Italy), for GJB2 gene mutations and for two deletions of the GJB6 gene (del GJB6 -D13S1830 and del GJB6 -D13S1854). We identified pathogenetic GJB2 mutations in 51 cases (15% of patients). No GJB6 mutation was found. We also examined the audiologic features of the patients for whom we had an etiologic diagnosis, in order to identify correlations between the severity of hearing loss and the type of mutation.
Assuntos
Conexinas/genética , Testes Genéticos , Perda Auditiva Neurossensorial/genética , Programas de Rastreamento/métodos , Mutação , Estimulação Acústica , Adolescente , Adulto , Audiometria , Percepção Auditiva , Criança , Pré-Escolar , Conexina 26 , Conexina 30 , Predisposição Genética para Doença , Perda Auditiva Neurossensorial/epidemiologia , Perda Auditiva Neurossensorial/fisiopatologia , Heterozigoto , Homozigoto , Humanos , Itália/epidemiologia , Pessoa de Meia-Idade , Fenótipo , Fatores de Risco , Índice de Gravidade de Doença , Adulto JovemRESUMO
In Drosophila, Pumilio (Pum) is important for neuronal homeostasis as well as learning and memory. We have recently characterized a mammalian homolog of Pum, Pum2, which is found in discrete RNA-containing particles in the somatodendritic compartment of polarized neurons. In this study, we investigated the role of Pum2 in developing and mature neurons by RNA interference. In immature neurons, loss of Pum2 led to enhanced dendritic outgrowth and arborization. In mature neurons, Pum2 down-regulation resulted in a significant reduction in dendritic spines and an increase in elongated dendritic filopodia. Furthermore, we observed an increase in excitatory synapse markers along dendritic shafts. Electrophysiological analysis of synaptic function of neurons lacking Pum2 revealed an increased miniature excitatory postsynaptic current frequency. We then identified two specific mRNAs coding for a known translational regulator, eIF4E, and for a voltage-gated sodium channel, Scn1a, which interacts with Pum2 in immunoprecipitations from brain lysates. Finally, we show that Pum2 regulates translation of the eIF4E mRNA. Taken together, our data reveal a previously undescribed role for Pum2 in dendrite morphogenesis, synapse function, and translational control.
Assuntos
Dendritos/fisiologia , Hipocampo/crescimento & desenvolvimento , Morfogênese/genética , Proteínas de Ligação a RNA/fisiologia , Sinapses/fisiologia , Animais , Ensaio de Desvio de Mobilidade Eletroforética , Eletrofisiologia , Fator de Iniciação 4E em Eucariotos/metabolismo , Hipocampo/citologia , Luciferases , Morfogênese/fisiologia , Canal de Sódio Disparado por Voltagem NAV1.1 , Proteínas do Tecido Nervoso/metabolismo , Interferência de RNA , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Sódio/metabolismoRESUMO
Progressive hearing loss is common in the human population, but we have few clues to the molecular basis. Mouse mutants with progressive hearing loss offer valuable insights, and ENU (N-ethyl-N-nitrosourea) mutagenesis is a useful way of generating models. We have characterised a new ENU-induced mouse mutant, Oblivion (allele symbol Obl), showing semi-dominant inheritance of hearing impairment. Obl/+ mutants showed increasing hearing impairment from post-natal day (P)20 to P90, and loss of auditory function was followed by a corresponding base to apex progression of hair cell degeneration. Obl/Obl mutants were small, showed severe vestibular dysfunction by 2 weeks of age, and were completely deaf from birth; sensory hair cells were completely degenerate in the basal turn of the cochlea, although hair cells appeared normal in the apex. We mapped the mutation to Chromosome 6. Mutation analysis of Atp2b2 showed a missense mutation (2630C-->T) in exon 15, causing a serine to phenylalanine substitution (S877F) in transmembrane domain 6 of the PMCA2 pump, the resident Ca(2+) pump of hair cell stereocilia. Transmembrane domain mutations in these pumps generally are believed to be incompatible with normal targeting of the protein to the plasma membrane. However, analyses of hair cells in cultured utricular maculae of Obl/Obl mice and of the mutant Obl pump in model cells showed that the protein was correctly targeted to the plasma membrane. Biochemical and biophysical characterisation showed that the pump had lost a significant portion of its non-stimulated Ca(2+) exporting ability. These findings can explain the progressive loss of auditory function, and indicate the limits in our ability to predict mechanism from sequence alone.
Assuntos
Cálcio/metabolismo , Surdez/genética , Células Ciliadas Auditivas/metabolismo , Mutação de Sentido Incorreto , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Equorina/metabolismo , Substituição de Aminoácidos , Animais , Membrana Celular/metabolismo , Células Cultivadas , Mapeamento Cromossômico , Análise Mutacional de DNA , Surdez/patologia , Surdez/fisiopatologia , Orelha Interna/patologia , Orelha Interna/ultraestrutura , Potenciais Evocados Auditivos do Tronco Encefálico , Células Ciliadas Auditivas/patologia , Camundongos , Microscopia Eletrônica de Varredura , Mutagênese , Sáculo e Utrículo/metabolismoRESUMO
Most important processes in cell life are regulated by calcium (Ca2+). A number of mechanisms have thus been developed to maintain the concentration of free Ca2+ inside cells at the level (100-200nM) necessary for the optimal operation of the targets of its regulatory function. The systems that move Ca2+ back and forth across membranes are important actors in its control. The plasma membrane calcium ATPase (PMCA pump) which ejects Ca2+ from all eukaryotic cell types will be the topic of this contribution. The pump uses a molecule of ATP to transport one molecule of Ca2+ from the cytosol to the external environment. It is a P-type ATPase encoded by four genes (ATP2B1-4), the transcripts of which undergo different types of alternative splicing. Many pump variants thus exist. Their multiplicity is best explained by the specific Ca2+ demands in different cell types. In keeping with these demands, the isoforms are differently expressed in tissues and cell types and have differential Ca2+ extruding properties. At very low Ca2+ concentrations the PMCAs are nearly inactive. They must be activated by calmodulin, by acid phospholipids, by protein kinases, and by other means, e.g., a dimerization process. Other proteins interact with the PMCAs (i.e., MAGUK and NHERF at the PDZ domain and calcineurin A in the main intracellular domain) to sort them to specific regions of the cell membrane or to regulate their function. In some cases the interaction is isoform, or even splice variant specific. PMCAs knock out (KO) mice have been generated and have contributed information on the importance of PMCAs to cells and organisms. So far, only one human genetic disease, hearing loss, has been traced back to a PMCA defect.
Assuntos
Membrana Celular/enzimologia , Modelos Moleculares , ATPases Transportadoras de Cálcio da Membrana Plasmática/fisiologia , Processamento Alternativo , Animais , Cálcio/metabolismo , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologiaRESUMO
A previous study has demonstrated that the ubiquitous plasma membrane Ca(2+) pump PMCA4 interacted with isoform epsilon of the 14-3-3 protein, whereas the nervous tissue-specific PMCA2 did not. The 14-3-3 proteins are widely expressed small acidic proteins, which modulate cell signaling, intracellular trafficking, transcription and apoptosis. The investigation has been extended to the other tissue-restricted pump (PMCA3) and to the other ubiquitous pump (PMCA1). At variance with PMCA2, PMCA3 interacted with the 14-3-3epsilon protein in a two-hybrid system assay, which could not be used for PMCA1. The 14-3-3epsilon protein immunoprecipitated with both PMCA3 and PMCA1 when expressed in HeLa cells. Pull-down experiments using GST-PMCA1 and GST-PMCA3 fusion products confirmed the interaction of both pumps with the 14-3-3epsilon protein. The binding was phosphorylation-independent with both PMCA3 and PMCA1. The 14-3-3zeta isoform also interacted with PMCA3; however, it did not interact with PMCA1. The effect of the interaction on the activity of the two pumps, and thus on the homeostasis of Ca(2+), was investigated by co-expressing the 14-3-3epsilon protein and PMCA3 or PMCA1 in CHO cells together with the recombinant Ca(2+) indicator aequorin: the ability of cells to re-establish the basal Ca(2+) concentration following a Ca(2+) transient induced by an InsP(3)-producing agonist was substantially decreased with both pumps, indicating that the interaction with the 14-3-3 protein inhibited the activity of both PMCA3 and PMCA1.
Assuntos
Proteínas 14-3-3/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Proteínas 14-3-3/efeitos dos fármacos , Proteínas 14-3-3/genética , Equorina , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Células CHO , Agonistas dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Células HeLa , Homeostase/efeitos dos fármacos , Homeostase/genética , Humanos , Indicadores e Reagentes , Dados de Sequência Molecular , Fosforilação , ATPases Transportadoras de Cálcio da Membrana Plasmática/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Isoformas de Proteínas/efeitos dos fármacos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
In mammals, four different genes encode four PMCA isoforms. PMCA1 and PMCA4 are expressed ubiquitously. PMCA2 and PMCA3 are expressed prevalently in the central nervous systems. More than 30 variants are generated by mechanisms of alternative splicing. The physiological meaning of the existence of such elevated number of isoforms is not clear, but it would be plausible to relate it to the cell-specific demands of Ca2+ homeostasis. To characterize functional specificity of PMCA variants we have investigated two aspects: the effects of the overexpression of the different PMCA variants on cellular Ca2+ handling and the existence of possible isoform-specific interactions with partner proteins using a yeast two-hybrid technique. The four basic PMCA isoforms were coexpressed in CHO cells together with the Ca2+-sensitive recombinant photoprotein aequorin. The effects of their overexpression on Ca2+ homeostasis were monitored in the living cells. They had revealed that the ubiquitous isoforms 1 and 4 are less effective in reducing the Ca2+ peaks generated by cell stimulation as compared to the neuron-specific isoforms 2 and 3. To establish whether these differences were related to different and new physiological regulators of the pump, the 90 N-terminal residues of PMCA2 and PMCA4 have been used as baits for the search of molecular partners. Screening of a human brain cDNA library with the PMCA4 bait specified the epsilon-isoform of protein 14-3-3, whereas no 14-3-3 epsilon clone was obtained with the PMCA2 bait. Overexpression of PMCA4/14-3-3 epsilon (but not of PMCA2/14-3-3 epsilon) in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca2+ was impaired. Thus, the interaction with 14-3-3 epsilon inhibited PMCA4 but not PMCA2. The role of PMCA2 has been further characterized by Ca2+ measurements in cells overexpressing different splicing variants. The results indicated that the combination of alternative splicing at two different sites in the pump structure was responsible for different functional characteristics of the pumps.
Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Isoformas de Proteínas/metabolismo , Animais , Células CHO , Membrana Celular/enzimologia , Cricetinae , Cricetulus , Humanos , Especificidade por SubstratoRESUMO
We ascertained a large Italian family with an autosomal dominant form of non-syndromic sensorineural hearing loss with vestibular involvement. A genome-wide scan found linkage to locus DFNA11. Sequencing of the MYO7A gene in the linked region identified a new missense mutation resulting in an Ala230Val change in the motor domain of the myosin VIIA. Myosin VIIA has already been implicated in several forms of deafness, but this is the third mutation causing a dominant form of deafness, located in the myosin VIIA motor domain in a region never involved in hearing loss until now. A modelled protein structure of myosin VII motor domain provides evidence for a significant functional effect of this missense mutation.