RESUMO
The process of neuronal differentiation is associated with neurite elongation and membrane biogenesis, and phosphatidylcholine (PtdCho) is the major membrane phospholipid in mammalian cells. During neuroblast differentiation, the transcription of two genes involved in PtdCho biosynthesis are stimulated: Chka gene for choline kinase (CK) alpha isoform and Pcyt1a gene for CTP:phosphocholine cytidylyltransferase (CCT) alpha isoform. Here we show that CKα is essential for neuronal differentiation. In addition, we demonstrated that KDM2B regulates CKα expression and, as a consequence, neuronal differentiation. This factor is up-regulated in the course of the neuroblasts proliferative and undifferentiated state and down-regulated during differentiation induced by retinoic acid (RA). During proliferation, KDM2B binds to the Box2 located in the Chka promoter repressing its transcription. Interestingly, KDM2B knockdown enhances the levels of CKα expression in neuroblast cells and induces neuronal differentiation even in the absence of RA. These results suggest that KDM2B is required for the appropriate regulation of CKα during neuronal differentiation and to the maintaining of the undifferentiated stage of neuroblast cells.
Assuntos
Colina Quinase/genética , Proteínas F-Box/metabolismo , Regulação Neoplásica da Expressão Gênica , Histona Desmetilases com o Domínio Jumonji/metabolismo , Neuroblastoma/genética , Tretinoína/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular Tumoral , Colina Quinase/metabolismo , Epigênese Genética , Proteínas F-Box/genética , Seguimentos , Técnicas de Silenciamento de Genes , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Células-Tronco Neurais/fisiologia , Neuroblastoma/mortalidade , Neuroblastoma/patologia , Prognóstico , Regiões Promotoras Genéticas/genética , RNA Interferente Pequeno/metabolismo , Regulação para CimaRESUMO
At low temperatures, Bacillus cereus synthesizes large amounts of unsaturated fatty acids (UFAs) with double bonds in positions Δ5 and Δ10, as well as Δ5,10 diunsaturated fatty acids. Through sequence homology searches, we identified two open reading frames (ORFs) encoding a putative Δ5 desaturase and a fatty acid acyl-lipid desaturase in the B. cereus ATCC 14579 genome, and these were named BC2983 and BC0400, respectively. Functional characterization of ORFs BC2983 and BC0400 by means of heterologous expression in Bacillus subtilis confirmed that they both encode acyl-lipid desaturases that use phospholipids as the substrates and have Δ5 and Δ10 desaturase activities. Thus, these ORFs were correspondingly named desA (Δ5 desaturase) and desB (Δ10 desaturase). We established that DesA utilizes ferredoxin and flavodoxins (Flds) as electron donors for the desaturation reaction, while DesB preferably employs Flds. In addition, increased amounts of UFAs were found when B. subtilis expressing B. cereus desaturases was subjected to a cold shock treatment, indicating that the activity or the expression of these enzymes is upregulated in response to a decrease in growth temperature. This represents the first work reporting the functional characterization of fatty acid desaturases from B. cereus.
Assuntos
Bacillus cereus/enzimologia , Bacillus cereus/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos Insaturados/biossíntese , Bacillus cereus/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Clonagem Molecular , Ácidos Graxos Dessaturases/genética , Ferredoxinas/metabolismo , Flavodoxina/metabolismo , Expressão Gênica , OxirreduçãoRESUMO
The Bacillus subtilis acyl lipid desaturase (Δ5-Des) is an iron-dependent integral membrane protein able to selectively introduce double bonds into long-chain fatty acids. In the last decade since its discovery, the molecular mechanism of Δ5-Des expression has been studied extensively. However, the mechanism of desaturation, which must rely on unknown bacterial proteins for electron transfer, has not yet been explored. The B. subtilis genome encodes three proteins that can act as potential electron donors of Δ5-Des, ferredoxin (Fer) and two flavodoxins (Flds) (YkuN and YkuP), which are encoded by the ykuNOP operon. Here we report that the disruption of either the fer gene or the ykuNOP operon decreases the desaturation of palmitic acid by â¼30%. Nevertheless, a fer ykuNOP mutant abolished the desaturation reaction almost completely. Our results establish Fer and the two Flds as redox partners for Δ5-Des and suggest that the Fer and Fld proteins could function physiologically in the biosynthesis of unsaturated fatty acids in B. subtilis. Although Flds have extensively been described as partners in a number of redox processes, this is the first report describing their role as electron donors in the fatty acid desaturation reaction.