RESUMO
Autosomal recessive conditions are a significant health burden with few treatments. Population carrier screening has been suggested as a means to tackle them. Little is known about the views of affected families despite the potential for direct impacts on them. Data are presented on attitudes among families affected by Spinal Muscular Atrophy (SMA) toward two population screening programs, pre-conception, and prenatal. Data were gathered through qualitative interviews (n = 36) and a survey (n = 337). Eighty-two survey participants had SMA and 255 were family members. The majority were in favor of screening (75%). Reasons for supporting pre-conception screening support were a belief that it would reduce SMA-related terminations and raise awareness of SMA in the population. For prenatal screening, reasons for support included a belief in the importance of informed decision-making and the need to reduce suffering. Key reasons for non-support of pre-conception screening included concerns about carrier stigmatization and social engineering. For prenatal screening, concerns focused on the collateral loss of high quality of life lives affected by SMA. This study highlights that those affected by SMA are predominantly in favor of screening, although pre-conception screening is most favored. While family members and adults with SMA had largely consistent views, perceptions varied according to the severity (type) of SMA, with those affected by SMA type II the least likely to support screening. These findings suggest that screening for SMA is a complex issue for affected families, underscoring the need to consider and include their views when planning and implementing screening programs. © 2016 Wiley Periodicals, Inc.
Assuntos
Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/epidemiologia , Vigilância da População , Adulto , Idade de Início , Família , Feminino , Dosagem de Genes , Aconselhamento Genético , Testes Genéticos , Humanos , Entrevistas como Assunto , Masculino , Programas de Rastreamento , Pessoa de Meia-Idade , Razão de Chances , Fenótipo , Cuidado Pré-Concepcional/métodos , Diagnóstico Pré-Natal/métodos , Índice de Gravidade de Doença , Inquéritos e Questionários , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
The childhood disorder spinal muscular atrophy (SMA) is caused by reduced expression of the survival motor neuron (SMN) protein. SMN is a multifunctional protein that has been implicated in the production, processing and transport of RNA and ribonucleoproteins (RNPs). Within the nucleus, SMN is predominantly targeted to Cajal bodies (CB), which are involved in the maturation and processing of several subclasses of RNPs. Here, we show that the SMN exon 2b-encoded domain (SMN2b) is independently sufficient to mediate CB targeting, but that the resulting bodies are less dynamic than those containing full-length SMN protein. We also show that while two SMN proteins harbouring SMA-causing point mutations (A2G and S262I) are efficiently targeted to CBs, they also display reduced nuclear movement.
Assuntos
Corpos Enovelados/metabolismo , Mutação , Proteínas do Complexo SMN/genética , Criança , Éxons , Células HeLa , Humanos , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Complexo SMN/metabolismoRESUMO
In response to immunogenic cell death inducers, calreticulin (CRT) translocates from its orthotopic localization in the lumen of the endoplasmic reticulum (ER) to the surface of the plasma membrane where it serves as an engulfment signal for antigen-presenting cells.(1) Here, we report that yet another ER protein, the lysyl-tRNA synthetase (KARS), was exposed on the surface of stressed cells, on which KARS co-localized with CRT in lipid rafts. Depletion of KARS with small interfering RNAs suppressed CRT exposure induced by anthracyclines or UVC light. In contrast to CRT, KARS was also found in the supernatant of stressed cells. Recombinant KARS protein was unable to influence the binding of recombinant CRT to the cell surface. Moreover, recombinant KARS protein was unable to stimulate macrophages in vitro. These results underscore the contribution of KARS to the emission of (one of) the principal signal(s) of immunogenic cell death, CRT exposure.
Assuntos
Calreticulina/metabolismo , Membrana Celular/metabolismo , Citotoxicidade Imunológica , Lisina-tRNA Ligase/metabolismo , Sequência de Aminoácidos , Antraciclinas/farmacologia , Morte Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Citotoxicidade Imunológica/efeitos dos fármacos , Células HeLa , Humanos , Lisina-tRNA Ligase/química , Dados de Sequência Molecular , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Childhood spinal muscular atrophy is caused by a reduced expression of the survival motor neuron (SMN) protein. SMN has been implicated in the axonal transport of beta-actin mRNA in both primary and transformed neuronal cell lines, and loss of this function could account, at least in part, for spinal muscular atrophy onset and pathological specificity. Here we have utilised a targeted screen to identify mRNA associated with SMN, Gemin2 and Gemin3 in the cytoplasm of a human neuroblastoma cell line, SHSY5Y. Importantly, we have provided the first direct evidence that beta-actin mRNA is present in SMN cytoplasmic complexes in SHSY5Y cells.
Assuntos
Actinas/genética , Citoplasma/metabolismo , Proteína DEAD-box 20/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Axônios , Linhagem Celular Tumoral , HumanosRESUMO
Calreticulin (CRT) is an endoplasmic reticulum (ER) chaperone responsible for glycoprotein folding and Ca(2+) homeostasis. CRT also has extracellular functions, e.g. tumor and apoptotic cell recognition and wound healing, but the mechanism of CRT extracellular release is unknown. Cytosolic localization of CRT is determined by signal peptide and subsequent retrotranslocation of CRT into the cytoplasm. Here, we show that under apoptotic stress conditions, the cytosolic concentration of CRT increases and associates with phosphatidylserine (PS) in a Ca(2)(+)-dependent manner. PS distribution is regulated by aminophospholipid translocase (APLT), which maintains PS on the cytosolic side of the cell membrane. APLT is sensitive to redox modifications of its SH groups by reactive nitrogen species. During apoptosis, both CRT expression and the concentration of nitric oxide (NO) increase. By using S-nitroso-l-cysteine-ethyl-ester, an intracellular NO donor and inhibitor of APLT, we showed that PS and CRT externalization occurred together in an S-nitrosothiol-dependent and caspase-independent manner. Furthermore, the CRT and PS are relocated as punctate clusters on the cell surface. Thus, CRT induced nitrosylation and its externalization with PS could explain how CRT acts as a bridging molecule during apoptotic cell clearance.
Assuntos
Apoptose , Calreticulina/metabolismo , Sequência de Bases , Primers do DNA , Células HeLa , Humanos , Células Jurkat , Microscopia de Fluorescência , Fosfatidilserinas/metabolismo , Reação em Cadeia da Polimerase , Frações Subcelulares/metabolismoRESUMO
The Ewing's sarcoma (EWS) protein is a ubiquitously expressed RNA chaperone. The EWS protein localizes predominantly to the nucleus. Previous reports have suggested that the EWS protein is capable of dimerizing. However, to date this has not been confirmed. Here, using a novel panel of recombinant proteins, we have performed an in vitro biomolecular interaction analysis of the EWS protein. We have demonstrated that all three arginine-glycine-glycine (RGG) motifs are capable of binding directly to the survival motor neuron protein, a Tudor domain containing EWS binding partner. We have also confirmed EWS is capable of self-associating, and we have mapped this binding domain to the RGG motifs. We have also found that self-association may be required for EWS nuclear import. This is the first direct evidence of RGG domains being involved in self-association and has implications on all RGG-containing proteins.
Assuntos
Proteínas do Tecido Nervoso/química , Multimerização Proteica , Proteína EWS de Ligação a RNA/química , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Arginina/metabolismo , Sítios de Ligação , Cromatografia em Gel , Glicina/metabolismo , Células HeLa , Humanos , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Ligação Proteica , Proteína EWS de Ligação a RNA/isolamento & purificação , Proteínas Recombinantes , Ressonância de Plasmônio de Superfície , Proteína 1 de Sobrevivência do Neurônio MotorRESUMO
Childhood spinal muscular atrophy (SMA) is caused by a reduction in survival motor neuron (SMN) protein. SMN is expressed in every cell type, but it is predominantly the lower motor neurones of the spinal cord that degenerate in SMA. SMN has been linked to the axonal transport of beta-actin mRNA, a breakdown in which could trigger disease onset. It is known that SMN is present in transport ribonucleoproteins (RNPs) granules that also contain Gemin2 and Gemin3. To further characterise these granules we have performed live cell imaging of GFP-tagged SMN, GFP-Gemin2, GFP-Gemin3, GFP-Gemin6 and GFP-Gemin7. In all, we have made two important observations: (1) SMN granules appear metamorphic; and (2) the SMN-Gemin complex(es) appears to localise to two distinct subsets of bodies in neurites; stationary bodies and smaller dynamic bodies. This study provides an insight into the neuronal function of the SMN complex.
Assuntos
Neuritos/metabolismo , Proteínas do Complexo SMN/metabolismo , Proteína 1 de Sobrevivência do Neurônio Motor/metabolismo , Vesículas Transportadoras/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/metabolismo , Humanos , Atrofias Musculares Espinais da Infância/metabolismoRESUMO
The Ewing Sarcoma (EWS) protein is a ubiquitously expressed RNA processing factor that localises predominantly to the nucleus. However, the mechanism through which EWS enters the nucleus remains unclear, with differing reports identifying three separate import signals within the EWS protein. Here we have utilized a panel of truncated EWS proteins to clarify the reported nuclear localisation signals. We describe three C-terminal domains that are important for efficient EWS nuclear localization: (1) the third RGG-motif; (2) the last 10 amino acids (known as the PY-import motif); and (3) the zinc-finger motif. Although these three domains are involved in nuclear import, they are not independently capable of driving the efficient import of a GFP-moiety. However, collectively they form a complex tripartite signal that efficiently drives GFP-import into the nucleus. This study helps clarify the EWS import signal, and the identification of the involvement of both the RGG- and zinc-finger motifs has wide reaching implications.
Assuntos
Neoplasias Ósseas/metabolismo , Núcleo Celular/metabolismo , Sinais de Localização Nuclear , Proteína EWS de Ligação a RNA/metabolismo , Sarcoma de Ewing/metabolismo , Dedos de Zinco , Transporte Ativo do Núcleo Celular , Citoplasma/metabolismo , Análise Mutacional de DNA , Humanos , Mutação , Sinais Direcionadores de Proteínas , Proteína EWS de Ligação a RNA/genéticaRESUMO
The childhood autosomal recessive disorder spinal muscular atrophy (SMA) is caused by mutations in the survival motor neuron (SMN) gene. SMN localizes diffusely in the cytoplasm and in distinct nuclear structures called Cajal bodies. Cajal bodies are believed to be the storage and processing sites of several ribonucleoproteins. Here, using a novel panel of SMN exon deletion constructs, we report a systematic analysis of internal targeting domains in the SMN protein. We demonstrate that the peptides encoded by exons 2b, 3 and 6 perform an integral role in the cellular targeting of SMN. In addition, we identify a nine amino acid motif within the highly conserved sequences of the exon 2b encoded domain that mediates Cajal body targeting and self-association. Deletion of this domain dramatically affects SMN activity and results in a dominant-negative clone. These results identify critical domains within the SMN protein and have an impact on our understanding of the SMN protein with regards to SMA as well as cellular biology.
Assuntos
Corpos Enovelados/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Atrofia Muscular Espinal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Corpos Enovelados/genética , Corpos Enovelados/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/química , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Éxons/genética , Células HeLa , Humanos , Imuno-Histoquímica , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas do Complexo SMN , Células Tumorais CultivadasRESUMO
The non-structural proteins NS1 and NS2 of the parvovirus minute virus of mice (MVM) are required for efficient virus replication. It has previously been shown that NS1 and NS2 interact and colocalize with the survival motor neuron (Smn) gene product in novel nuclear structures that are formed late in infection, termed Smn-associated APAR (autonomous parvovirus-associated replication) bodies (SAABs). It is not clear what molecular viral intermediate(s) contribute to SAAB formation. The current results address the role of NS2 in SAAB formation. In highly synchronized wild-type MVM infection of murine A9(2L) cells, NS2 colocalizes with Smn and other SAAB constituents. An MVM mutant that does not produce NS2 still generates SAABS, albeit with a temporal delay. The lag in SAAB formation seen in the absence of NS2 is probably related to the temporal delay in virus replication, suggesting that, whilst NS2 is required for efficient viral infection, it is dispensable for SAAB formation.
Assuntos
Corpos Enovelados/metabolismo , Vírus Miúdo do Camundongo/fisiologia , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Autoantígenos , Linhagem Celular , Regulação Viral da Expressão Gênica , Camundongos , Vírus Miúdo do Camundongo/patogenicidade , Replicação Viral , Proteínas Centrais de snRNPRESUMO
Spinal muscular atrophy (SMA) is caused by homozygous loss of the survival motor neuron (SMN1) gene. In virtually all SMA patients, a nearly identical copy gene is present, SMN2. SMN2 cannot fully compensate for the loss of SMN1 because the majority of transcripts derived from SMN2 lack a critical exon (exon 7), resulting in a dysfunctional SMN protein. Therefore, the critical distinction between a functional and a dysfunctional SMN protein is the inclusion or the exclusion of the exon 7 encoded peptide. To determine the role of the 16 amino acids encoded by SMN exon 7, a panel of synthetic mutations were transiently expressed in SMA patient fibroblasts and HeLa cells. Consistent with previous reports, the protein encoded by SMN exons 1-6 was primarily restricted to the nucleus. However, a variety of heterologous sequences fused to the C-terminus of SMN exons 1-6 allowed mutant SMN proteins to properly distribute to the cytoplasm and to the nuclear gems. These data demonstrate that the SMN exon 7 sequence is not specifically required, rather this region functions as a non-specific 'tail' that facilitates proper localization. Therefore, a possible means to restore additional activity to the SMNDelta7 protein could be to induce a longer C-terminus by suppressing recognition of the native stop codon. To address this possibility, aminoglycosides were examined for their ability to restore detectable levels of SMN protein in SMA patient fibroblasts. Aminoglycosides can suppress the accurate identification of translation termination codons in eukaryotic cells. Consistent with this, treatment of SMA patient fibroblasts with tobramycin and amikacin resulted in a quantitative increase in SMN-positive gems and an overall increase in detectable SMN protein. Taken together, this work describes the role of the critical exon 7 region and identifies a possible alternative approach for therapeutic intervention.
Assuntos
Aminoglicosídeos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Alanina/metabolismo , Amicacina/farmacologia , Substituição de Aminoácidos , Aminoglicosídeos/farmacologia , Antibacterianos/farmacologia , Anticorpos Monoclonais/metabolismo , Western Blotting , Células Cultivadas , Códon de Terminação , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Éxons , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fluoresceína-5-Isotiocianato , Imunofluorescência , Corantes Fluorescentes , Deleção de Genes , Células HeLa , Homozigoto , Humanos , Imuno-Histoquímica , Indóis , Cinética , Proteínas do Tecido Nervoso/deficiência , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor , Distribuição Tecidual , Tobramicina/farmacologia , Transcrição GênicaRESUMO
Consumption of red meat is associated with an increased risk of colorectal cancer, whereas cruciferous vegetable consumption reduces cancer risk. While the mechanisms remain to be determined, cruciferous vegetables may act by altering the metabolism of carcinogens present in cooked food, such as the heterocyclic amine 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). The aim of this study was to evaluate the effect of cruciferous vegetable consumption on the metabolism of PhIP in 20 non-smoking Caucasian male subjects. The study consisted of three 12-day phases, namely two periods of avoidance of cruciferous vegetables (phases 1 and 3) and a high cruciferous vegetable diet period (phase 2), when subjects ingested 250 g each of Brussels sprouts and broccoli per day. At the end of each study phase, the subjects consumed a cooked meat meal containing 4.90 microg PhIP and urine samples were collected for up to 48 h. Cruciferous vegetable consumption significantly increased hepatic CYP1A2, as demonstrated by changes in saliva caffeine kinetics. Samples of N(2)-hydroxy-PhIP-N(2)-glucuronide (the major urinary metabolite of PhIP in humans), N(2)-hydroxy-PhIP-N(3)-glucuronide and their trideuterated derivatives (to serve as internal standards) were synthesized and a liquid chromatography-mass spectrometry-mass spectrometry method developed for their analysis. In phases 1 and 3, the excretion of N(2)-hydroxy-N(2)-PhIP-glucuronide in 0-48 h urine samples was six times that of N(2)-hydroxy-PhIP-N(3)-glucuronide. Cruciferous vegetable consumption significantly increased the urinary excretion of N(2)-hydroxy-PhIP-N(2)-glucuronide in 0-48 h urine samples to 127 and 136% of levels observed in phases 1 and 3, respectively. In contrast, the urinary excretion of N(2)-hydroxy-PhIP-N(3)-glucuronide was unchanged. While the urinary excretion of both PhIP metabolites accounted for approximately 39% of the PhIP dose in phases 1 and 3, they accounted for approximately 49% of the dose in phase 2. This study demonstrates that cruciferous vegetable consumption can induce both the phase I and II metabolism of PhIP in humans.
Assuntos
Biomarcadores/urina , Brassica/metabolismo , Carcinógenos/metabolismo , Dieta , Imidazóis/metabolismo , Verduras , Cromatografia Líquida , Citocromo P-450 CYP1A2/metabolismo , Glucuronídeos/urina , Glucuronosiltransferase/metabolismo , Humanos , Fígado/enzimologia , Masculino , Espectrometria de Massas , População BrancaRESUMO
The aim of this study was to investigate xenobiotic metabolism and induction of cytochrome P450 (CYP) forms in precision-cut rat liver and lung slices, employing nicotine as a model compound. Freshly cut rat liver and lung slices metabolised nicotine to the major metabolite cotinine. Observed Km values for cotinine formation in liver and lung slices were 323 and 41.7 microM, respectively, with corresponding V(max) values of 47.2 and 3.21 pmol/min/mg protein, respectively. Rat liver and lung slices were cultured for 48 h with Aroclor 1254, benzo(a)pyrene, nicotine and cotinine. Both Aroclor 1254 and benzo(a)pyrene produced a marked induction of CYP1A-dependent 7-ethoxyresorufin O-deethylase activity in both liver and lung slices. However, while nicotine induced 7-ethoxyresorufin O-deethylase activity in lung slices, but not in liver slices, cotinine did not induce enzyme activity in either liver or lung slices. Overall, while higher rates of nicotine metabolism were observed in rat liver slices, nicotine-induced CYP1A form induction was observed in lung slices. These results demonstrate the usefulness of precision-cut tissue slices for studying tissue differences in xenobiotic metabolism and CYP form induction.
Assuntos
Sistema Enzimático do Citocromo P-450/biossíntese , Fígado/metabolismo , Pulmão/metabolismo , Nicotina/metabolismo , Agonistas Nicotínicos/metabolismo , Animais , Células Cultivadas , Citocromo P-450 CYP1A1/metabolismo , Indução Enzimática/efeitos dos fármacos , Técnicas In Vitro , Isoenzimas/biossíntese , Cinética , Fígado/enzimologia , Pulmão/enzimologia , Masculino , Bifenilos Policlorados/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Spinal muscular atrophy (SMA) is a degenerative disorder of spinal motor neurons caused by homozygous mutations in the survival motor neuron (SMN1) gene. Because increased tissue levels of human SMN protein (hSMN) in transgenic mice reduce the motor neuron loss caused by murine SMN knockout, we engineered a recombinant SMN fusion protein to deliver exogenous hSMN to the cytosolic compartment of motor neurons. The fusion protein, SDT, is comprised of hSMN linked to the catalytic and transmembrane domains of diphtheria toxin (DTx) followed by fragment C of tetanus toxin (TTC). Following overexpression in Escherichia coli, SDT possessed a subunit molecular weight of approximately 130 kDa as revealed by both SDS-PAGE and immunoblot analyses with anti-SMN, anti-DTx, and anti-TTC antibodies. Like wild-type SMN, purified SDT showed specific binding in vitro to an RG peptide derived from Ewing's sarcoma protein. The fusion protein also bound to cultured primary neurons in amounts similar to those achieved by TTC. Unlike the case with TTC, however, immunolabeling of SDT-treated neurons with anti-TTC and anti-SMN antibodies showed staining restricted to the cell surface. Results from cytotoxicity studies in which the DTx catalytic domain of SDT was used as a reporter protein for internalization and membrane translocation activity suggest that the SMN moiety of the fusion protein is interfering with one or both of these processes. While these studies indicate that SDT may not be useful for SMA therapy, the use of the TTC:DTx fusion construct to deliver other passenger proteins to the neuronal cytosol should not be ruled out.
Assuntos
Atrofia Muscular Espinal/tratamento farmacológico , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/genética , Proteínas Recombinantes de Fusão/farmacologia , Toxina Tetânica/genética , Animais , Animais Recém-Nascidos , Anticorpos/imunologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Citotoxinas/genética , Citotoxinas/imunologia , Citotoxinas/farmacologia , Toxina Diftérica/genética , Toxina Diftérica/imunologia , Relação Dose-Resposta a Droga , Endocitose/imunologia , Imuno-Histoquímica , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Proteínas do Tecido Nervoso/imunologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/imunologia , Ligação Proteica/imunologia , Estrutura Terciária de Proteína/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/imunologia , Proteínas de Ligação a RNA , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor , Toxina Tetânica/imunologiaRESUMO
The survival motor neuron (SMN) gene is the spinal muscular atrophy (SMA) determining gene. Here we report that the SMN protein product interacts in vitro and in vivo with the arginine/glycine (RG)-rich RNA binding protein and transcription factor, Ewing's sarcoma (EWS). Recently, the SMN encoded Tudor domain (exon 3) and the YG-motifs (exon 6) have been shown to be involved in binding to RG-rich proteins. Here, we demonstrate that the Tudor domain encoded by SMN exon 3 is independently sufficient to mediate the interaction with EWS. Synthetic mutations within the Tudor domain, as well as a SMA patient-derived mutation within exon 3, reduced the levels of the SMN/EWS interaction. Carboxyl-terminal SMN mutations that prevent formation of SMN oligomers also indirectly reduced EWS binding. A role for arginine methylation has been observed in some RG-containing SMN-interacting proteins. Here we demonstrate that SMN interacts with non-methylated EWS and an EWS-derived RG-containing peptide. In contrast to previously reported results, symmetrical dimethylation of the EWS-derived RG-peptide results in a quantitative increase in the dissociation rate between SMN and the symmetrical dimethylated EWS RG-peptide. Consistent with the interaction data, endogenous and transiently expressed SMN co-localizes with endogenous EWS in a number of cultured cell lines, as well as rat primary neuron cultures. Anti-sense RNA experiments, however, demonstrate that EWS does not mediate the nuclear distribution of SMN or other Cajal body components.
Assuntos
Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , Proteína EWS de Ligação a RNA/genética , Animais , Arginina/metabolismo , Sítios de Ligação/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Éxons/genética , Humanos , Metilação , Atrofia Muscular Espinal/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Peptídeos/metabolismo , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , RNA Antissenso , Proteína EWS de Ligação a RNA/química , Proteína EWS de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA , Ratos , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio MotorRESUMO
The metabolism of two thiofurans, namely furfuryl mercaptan (FM) and 2-methyl-3-furanthiol (MTF), to their corresponding methyl sulphide and methyl sulphoxide derivatives has been studied in male Sprague-Dawley rat hepatocytes and liver microsomes. Rat hepatocytes converted FM to furfuryl methyl sulphoxide (FMSO) and MTF to 2-methyl-3-(methylthio)furan sulphoxide (MMFSO). Liver microsomes catalysed the NADPH-dependent metabolism of furfuryl methyl sulphide (FMS) to FMSO and 2-methyl-3-(methylthio)furan sulphide (MMFS) to MMFSO. FMS and MMFS metabolism to their thiofuran methyl sulphoxide derivatives was induced by the treatment of rats with Aroclor 1254 and inhibited in liver microsomes treated with 1-aminobenzotriazole. The NADPH-dependent metabolism of FM to FMSO and MTF to MMFSO in liver microsomes was observed in the presence of S-adenosylmethionine. In summary, both thiofurans can be metabolised in rat liver to their thiofuran methyl sulphide derivatives which can be subsequently S-oxidised to form thiofuran methyl sulphoxides. FM and MTF appear to be substrates for rat hepatic microsomal thiol methyltransferase and the S-oxidation of FMS and MMFS appears to be primarily catalysed by cytochrome P450 forms.
Assuntos
Furanos/metabolismo , Fígado/metabolismo , Compostos de Sulfidrila/metabolismo , Algoritmos , Animais , Sistema Enzimático do Citocromo P-450/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Cinética , Masculino , Espectrometria de Massas , Metiltransferases/metabolismo , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Monoaminoxidase/metabolismo , NADH NADPH Oxirredutases/metabolismo , Ratos , Ratos Sprague-Dawley , Triazóis/farmacologiaRESUMO
Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by mutations in the survival motor neuron gene (SMN1). While it has been shown that the SMN protein is involved in spliceosome biogenesis and pre-mRNA splicing, there is increasing evidence indicating that SMN may also perform important functions in the nucleolus. We demonstrate here through the use of a previously characterized polyclonal anti-SMN antibody, abSMN, that the SMN protein shows a striking colocalization with the nucleolar protein, fibrillarin, in both nucleoli and Cajal bodies/gems of primary neurons. Immunoblot analysis with antifibrillarin and two different anti-SMN antibodies reveals that SMN and fibrillarin also cofractionate in the insoluble protein fraction of cultured cell lysates. Immunoprecipitation experiments using whole cell extracts of HeLa cells and cultured neurons revealed that abSMN coprecipitated small amounts of the U3 small nucleolar RNA (snoRNA) previously shown to be associated with fibrillarin in vivo. These studies raise the possibility that SMN may serve a function in rRNA maturation/ribosome synthesis similar to its role in spliceosome biogenesis.
Assuntos
Nucléolo Celular/metabolismo , Neurônios Motores/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Northern Blotting , Western Blotting , Neoplasias Encefálicas/metabolismo , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Camundongos , Microscopia de Fluorescência , Neurônios Motores/ultraestrutura , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Neuroblastoma/metabolismo , Testes de Precipitina , Proteínas de Ligação a RNA , Coelhos , Ratos , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Proteínas do Complexo SMN , Proteína 1 de Sobrevivência do Neurônio Motor , Células Tumorais CultivadasRESUMO
The human survival motor neuron (SMN) gene is the spinal muscular atrophy-determining gene, and a knockout of the murine Smn gene results in preembryonic lethality. Here we show that SMN can directly interact in vitro and in vivo with the large nonstructural protein NS1 of the autonomous parvovirus minute virus of mice (MVM), a protein essential for viral replication and a potent transcriptional activator. Typically, SMN localizes within nuclear Cajal bodies and diffusely in the cytoplasm. Following transient NS1expression, SMN and NS1 colocalize within Cajal bodies. At early time points following parvovirus infection, NS1 fails to colocalize with SMN within Cajal bodies; however, during the course of MVM infection, dramatic nuclear alterations occur. Formerly distinct nuclear bodies such as Cajal bodies, promyelocytic leukemia gene product (PML) oncogenic domains (PODs), speckles, and autonomous parvovirus-associated replication (APAR) bodies are seen aggregating at later points in infection. These newly formed large nuclear bodies (termed SMN-associated APAR bodies) are active sites of viral replication and viral capsid assembly. These results highlight the transient nature of nuclear bodies and their contents and identify a novel nuclear body formed during infection. Furthermore, simple transient expression of the viral nonstructural proteins is insufficient to induce this nuclear reorganization, suggesting that this event is induced specifically by a step in the viral infection process.
Assuntos
Corpos Enovelados/metabolismo , Vírus Miúdo do Camundongo/patogenicidade , Proteínas do Tecido Nervoso/metabolismo , Infecções por Parvoviridae/virologia , Proteínas não Estruturais Virais/metabolismo , Animais , Técnicas Biossensoriais , Linhagem Celular , Corpos Enovelados/virologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Humanos , Imuno-Histoquímica , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas de Ligação a RNA , Proteínas do Complexo SMN , Proteínas não Estruturais Virais/genéticaRESUMO
Proximal spinal muscular atrophy (SMA) is caused by the homozygous loss of survival motor neuron (SMN1). SMN2, a nearly identical copy gene, is present in all SMA patients; however this gene cannot provide protection from disease due to the aberrant splicing of a critical exon. SMN1-derived transcripts are exclusively full-length, whereas SMN2-derived transcripts predominantly lack SMN exon 7. A single non-polymorphic nucleotide difference (C in SMN1; T in SMN2) is responsible for the alternative splicing patterns. We have previously shown that transient expression of an SR-like splicing factor, hTra2 beta 1, stimulates inclusion of exon 7 in SMN2-derived mini-gene transcripts through an interaction with the AG-rich exonic splice enhancer within exon 7. We now demonstrate that a second splicing factor, SRp30c, can stimulate SMN exon 7-inclusion and that this activity required the same AG-rich enhancer as hTra2 beta 1. SRp30c did not directly associate with SMN exon 7; rather its association with the exonic enhancer was mediated by a direct interaction with hTra2 beta 1. In the absence of the hTra2 beta 1 binding site, SRp30c failed to complex with SMN exon 7. Taken together, these results identify SRp30c as a modulator of SMN exon 7-inclusion and provide insight into the molecular regulation of this critical exon.
Assuntos
Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas de Ligação a RNA , Processamento Alternativo , Sequência de Bases , Sítios de Ligação , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Elementos Facilitadores Genéticos/genética , Éxons , Células HeLa , Humanos , Atrofia Muscular Espinal/metabolismo , Isoformas de Proteínas/genética , RNA/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas do Complexo SMN , Fatores de Processamento de Serina-Arginina , Proteína 1 de Sobrevivência do Neurônio Motor , Proteína 2 de Sobrevivência do Neurônio Motor , Transcrição Gênica , Células Tumorais CultivadasRESUMO
Mutations in the SMN1 (survival motor neuron 1) gene cause spinal muscular atrophy (SMA). We now show that SMN protein, the SMN1 gene product, interacts directly with the tumor suppressor protein, p53. Pathogenic missense mutations in SMN reduce both self-association and p53 binding by SMN, and the extent of the reductions correlate with disease severity. The inactive, truncated form of SMN produced by the SMN2 gene in SMA patients fails to bind p53 efficiently. SMN and p53 co-localize in nuclear Cajal bodies, but p53 redistributes to the nucleolus in fibroblasts from SMA patients. These results suggest a functional interaction between SMN and p53, and the potential for apoptosis when this interaction is impaired may explain motor neuron death in SMA.