RESUMO
Myosin Va (MyoVa) is an actin-based molecular motor that plays key roles in the final stages of secretory pathways, including neurotransmitter release. Several studies have addressed how MyoVa coordinates the trafficking of secretory vesicles, but why this molecular motor is found in exosomes is still unclear. In this work, using a yeast two-hybrid screening system, we identified the direct interaction between the globular tail domain (GTD) of MyoVa and four protein components of exosomes: the WD repeat-containing protein 48 (WDR48), the cold shock domain-containing protein E1 (CSDE1), the tandem C2 domain-containing protein 1 (TC2N), and the enzyme spermine synthase (SMS). The interaction between the GTD of MyoVa and SMS was further validated in vitro and displayed a Kd in the low micromolar range (3.5 ± 0.5 µM). SMS localized together with MyoVa in cytoplasmic vesicles of breast cancer MCF-7 and neuroblastoma SH-SY5Y cell lines, known to produce exosomes. Moreover, MYO5A knockdown decreased the expression of SMS gene and rendered the distribution of SMS protein diffuse, supporting a role for MyoVa in SMS expression and targeting.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Exossomos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Espermina Sintase/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Células Cultivadas , Exossomos/genética , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Células MCF-7 , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Ligação Proteica , Transporte Proteico , Interferência de RNA , Espermina Sintase/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Snyder-Robinson syndrome, also known as spermine synthase deficiency, is an X-linked intellectual disability syndrome (OMIM #390583). First described by Drs. Snyder and Robinson in 1969, this syndrome is characterized by an asthenic body habitus, facial dysmorphism, broad-based gait, and osteoporosis with frequent fractures. We report here a pediatric autopsy of a 4 year old male with a history of intellectual disability, gait abnormalities, multiple fractures, and seizures previously diagnosed with Snyder-Robinson syndrome with an SMS gene mutation (c.831G>T:p.L277F). The cause of death was hypoxic-ischemic encephalopathy secondary to prolonged seizure activity. Although Snyder-Robinson syndrome is rare, the need to recognize clinical findings in order to trigger genetic testing has likely resulted in under diagnosis.
Assuntos
Humanos , Masculino , Pré-Escolar , Deficiência Intelectual Ligada ao Cromossomo X/patologia , Autopsia , Evolução Fatal , Hipóxia-Isquemia Encefálica/patologia , Deficiência Intelectual/patologia , Deficiência Intelectual Ligada ao Cromossomo X/diagnóstico , Convulsões/patologia , Espermina SintaseRESUMO
Polyamines are ubiquitous positively charged metabolites that play an important role in wide fundamental cellular processes; because of their importance, the homeostasis of these amines is tightly regulated. Spermine synthase catalyzes the formation of polyamine spermine, which is necessary for growth and development in higher eukaryotes. Previously, we reported a stress inducible spermine synthase 1 (ZmSPMS1) gene from maize. The ZmSPMS1 enzyme differs from their dicot orthologous by a C-terminal extension, which contains a degradation PEST sequence involved in its turnover. Herein, we demonstrate that ZmSPMS1 protein interacts with itself in split yeast two-hybrid (Y2H) assays. A Bimolecular Fluorescence Complementation (BiFC) assay revealed that ZmSPMS1 homodimer has a cytoplasmic localization. In order to gain a better understanding about ZmSPMS1 interaction, two deletion constructs of ZmSPMS1 protein were obtained. The ΔN-ZmSPMS1 version, where the first 74 N-terminal amino acids were eliminated, showed reduced capability of dimer formation, whereas the ΔC-ZmSPMS1 version, lacking the last 40 C-terminal residues, dramatically abated the ZmSPMS1-ZmSPMS1 protein interaction. Recombinant protein expression in Escherichia coli of ZmSPMS1 derived versions revealed that deletion of its N-terminal domain affected the spermine biosynthesis, whereas C-terminal ZmSPMS1 truncated version fail to generate this polyamine. These data suggest that N- and C-terminal domains of ZmSPMS1 play a role in a functional homodimer.
Assuntos
Espaço Intracelular/metabolismo , Multimerização Proteica , Espermina Sintase/metabolismo , Zea mays/enzimologia , Fluorescência , Folhas de Planta/metabolismo , Poliaminas/metabolismo , Ligação Proteica , Nicotiana/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina/metabolismoRESUMO
Polyamines are low molecular weight aliphatic compounds involved in various biochemical, cellular and physiological processes in all organisms. In plants, genes involved in polyamine biosynthesis and catabolism are regulated at transcriptional, translational, and posttranslational level. In this research, we focused on the characterization of a PEST sequence (rich in proline, glutamic acid, serine, and threonine) of the maize spermine synthase 1 (ZmSPMS1). To this aim, 123 bp encoding 40 amino acids of the C-terminal region of the ZmSPMS1 enzyme containing the PEST sequence were fused to the GUS reporter gene. This fusion was evaluated in Arabidopsis thaliana transgenic lines and onion monolayers transient expression system. The ZmSPMS1 PEST sequence leads to specific degradation of the GUS reporter protein. It is suggested that the 26S proteasome may be involved in GUS::PEST fusion degradation in both onion and Arabidopsis. The PEST sequences appear to be present in plant spermine synthases, mainly in monocots.
Assuntos
Glucuronidase/metabolismo , Proteínas de Plantas/metabolismo , Proteólise , Espermina Sintase/metabolismo , Zea mays/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Western Blotting , Inibidores de Cisteína Proteinase/farmacologia , Genes de Plantas/genética , Glucuronidase/genética , Leupeptinas/farmacologia , Dados de Sequência Molecular , Cebolas/citologia , Cebolas/genética , Cebolas/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espermina Sintase/genética , Zea mays/genéticaRESUMO
Polyamines play important roles in cell physiology including effects on the structure of cellular macromolecules, gene expression, protein function, nucleic acid and protein synthesis, regulation of ion channels, and providing protection from oxidative damage. Vertebrates contain two polyamines, spermidine and spermine, as well as their precursor, the diamine putrescine. Although spermidine has an essential and unique role as the precursor of hypusine a post-translational modification of the elongation factor eIF5A, which is necessary for this protein to function in protein synthesis, no unique role for spermine has been identified unequivocally. The existence of a discrete spermine synthase enzyme that converts spermidine to spermine suggest that spermine must be needed and this is confirmed by studies with Gy mice and human patients with Snyder-Robinson syndrome in which spermine synthase is absent or greatly reduced. In both cases, this leads to a severe phenotype with multiple effects among which are intellectual disability, other neurological changes, hypotonia, and reduced growth of muscle and bone. This review describes these alterations and focuses on the roles of spermine which may contribute to these phenotypes including reducing damage due to reactive oxygen species, protection from stress, permitting correct current flow through inwardly rectifying K(+) channels, controlling activity of brain glutamate receptors involved in learning and memory, and affecting growth responses. Additional possibilities include acting as storage reservoir for maintaining appropriate levels of free spermidine and a possible non-catalytic role for spermine synthase protein.
Assuntos
Deficiência Intelectual Ligada ao Cromossomo X/metabolismo , Deficiência Intelectual Ligada ao Cromossomo X/patologia , Espermina/metabolismo , Animais , Humanos , Camundongos , Espermina Sintase/metabolismoRESUMO
Polyamines (putrescine, spermidine and spermine) are traditionally implicated in the response of plants to environmental cues. Free spermine accumulation has been suggested as a particular feature of long-term salt stress, and in the model plant Arabidopsis thaliana the spermine synthase gene (AtSPMS) has been reported as inducible by abscisic acid (ABA) and acute salt stress treatments. With the aim to unravel the physiological role of free spermine during salinity, we analyzed polyamine metabolism in A. thaliana salt-hypersensitive sos mutants (salt overlay sensitive; sos1-1, sos2-1 and sos3-1), and studied the salt stress tolerance of the mutants in spermine and thermospermine synthesis (acl5-1, spms-1 and acl5-1/spms-1). Results presented here indicate that induction in polyamine metabolism is a SOS-independent response to salinity and is globally over-induced in a sensitive background. In addition, under long-term salinity, the mutants in the synthesis of spermine and thermospermine (acl5-1, spms-1 and double acl5-1/spms-1) accumulated more Na(+) and performed worst than WT in survival experiments. Therefore, support is given to a role for these higher polyamines in salt tolerance mechanisms.
Assuntos
Arabidopsis/metabolismo , Tolerância ao Sal/fisiologia , Espermina/metabolismo , Arabidopsis/genética , Poliaminas Biogênicas/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/efeitos dos fármacos , Variação Genética , Reguladores de Crescimento de Plantas/metabolismo , Salinidade , Tolerância ao Sal/genética , Cloreto de Sódio/metabolismo , Espermina/análogos & derivados , Espermina/biossíntese , Espermina Sintase/genética , Espermina Sintase/metabolismoRESUMO
The role of the tetraamine spermine in plant defense against pathogens was investigated by using the Arabidopsis (Arabidopsis thaliana)-Pseudomonas viridiflava pathosystem. The effects of perturbations of plant spermine levels on susceptibility to bacterial infection were evaluated in transgenic plants (35S::spermine synthase [SPMS]) that overexpressed the SPMS gene and accumulated spermine, as well as in spms mutants with low spermine levels. The former exhibited higher resistance to P. viridiflava than wild-type plants, while the latter were more susceptible. Exogenous supply of spermine to wild-type plants also increased disease resistance. Increased resistance provided by spermine was partly counteracted by the polyamine oxidase inhibitor SL-11061, demonstrating that the protective effect of spermine partly depends on its oxidation. In addition, global changes in gene expression resulting from perturbations of spermine levels were analyzed by transcript profiling 35S::SPMS-9 and spms-2 plants. Overexpression of 602 genes was detected in 35S::SPMS-9 plants, while 312 genes were down-regulated, as compared to the wild type. In the spms-2 line, 211 and 158 genes were up- and down-regulated, respectively. Analysis of gene ontology term enrichment demonstrated that many genes overexpressed only in 35S::SPMS-9 participate in pathogen perception and defense responses. Notably, several families of disease resistance genes, transcription factors, kinases, and nucleotide- and DNA/RNA-binding proteins were overexpressed in this line. Thus, a number of spermine-responsive genes potentially involved in resistance to P. viridiflava were identified. The obtained results support the idea that spermine contributes to plant resistance to P. viridiflava.
Assuntos
Arabidopsis/imunologia , Arabidopsis/microbiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Pseudomonas/fisiologia , Espermina Sintase/genética , Espermina/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Contagem de Colônia Microbiana , Regulação Enzimológica da Expressão Gênica , Genes de Plantas/genética , Mutação/genética , Oxirredução , Plantas Geneticamente Modificadas , Pseudomonas/crescimento & desenvolvimento , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espermina Sintase/metabolismo , Fatores de Tempo , Transcrição GênicaRESUMO
Snyder-Robinson syndrome (SRS, OMIM 309583) is a rare X-linked syndrome characterized by mental retardation, marfanoid habitus, skeletal defects, osteoporosis, and facial asymmetry. Linkage analysis localized the related gene to Xp21.3-p22.12, and a G-to-A transition at point +5 of intron 4 of the spermine synthase gene, which caused truncation of the SMS protein and loss of enzyme activity, was identified in the original family. Here we describe another family with Snyder-Robinson syndrome in two Mexican brothers and a novel mutation (c.496T>G) in the exon 5 of the SMS gene confirming its involvement in this rare X-linked mental retardation syndrome.
Assuntos
Cromossomos Humanos X , Genes Ligados ao Cromossomo X , Deficiência Intelectual Ligada ao Cromossomo X/genética , Mutação de Sentido Incorreto , Espermina Sintase/genética , Adulto , Análise Mutacional de DNA , Éxons , Ligação Genética , Humanos , Deficiência Intelectual/genética , Masculino , Síndrome de Marfan/genética , Osteoporose/genética , Linhagem , Escoliose/genética , Irmãos , Adulto JovemRESUMO
2,4-Dichlorophenoxyacetic acid (2,4-D) is a herbicide extensively used in agriculture. It was considered of interest to study its toxicity on animal cells. We had previously determined that 1 mM 2,4-D can inhibit cell growth, DNA and protein synthesis of cultured Chinese hamster ovary cells (CHO) with cell accumulation in the G1/S interphase of the cell cycle. The present work examined the effects of 2,4-D on polyamine biosynthesis. The results suggest some possible mechanism of the herbicide's toxic effects on animal cells.