RESUMO
The marine diatom Phaeodactylum tricornutum, a widely used forage species, has a storage lipid content of up to 30% dry cell weight. To explore the mechanism behind the high storage lipid accumulation in this diatom, acetyl-CoA carboxylase (ACCase), which catalyzes the first committed step of the fatty acid biosynthetic pathway, was characterized in this study. A homogeneous type of ACCase (PtACC) was identified from P. tricornutum by homology searches. The first exon of the ACCase gene (PtACC-1) was cloned. PtACC-1 was fused with a Myc epitope tag and cloned into plasmid pMD18 driven by the LacZ promoter and expressed in Escherichia coli. The expression of the PtACC-1-Myc protein was verified by Western blot. The neutral lipid content in transformed E. coli increased substantially by twofold as determined by Nile red fluorescent dye staining. Concomitantly, ACCase activity increased by 1.72-fold. The fatty acid composition, analyzed by GC-MS, demonstrated a significant difference in the ratio of saturated fatty acids and monounsaturated fatty acids (MUFAs). MUFAs of PtACC-1 expressing cells increased by 13%. This study represents the first characterization of the key domains of ACCase from a diatom and demonstrates high neutral lipid accumulation in E. coli expressing PtACC-1, providing an additional genetic resource with the potential for biodiesel development.
Assuntos
Diatomáceas/genética , Escherichia coli/genética , Ácidos Graxos/metabolismo , Subunidades Proteicas/genética , Acetil-CoA Carboxilase , Clonagem Molecular/métodos , Diatomáceas/metabolismo , Escherichia coli/metabolismo , Éxons/genética , Ácidos Graxos/genética , Expressão Gênica/genética , Filogenia , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/metabolismoRESUMO
Oxsterol binding protein-related protein 4 (ORP4) is essential for cell proliferation, but the underlying mechanism is unclear. ORP4 is expressed as three variants, ORP4L, ORP4M and ORP4S. Here, we reported that silencing of ORP4L with specific small interfering RNA (siRNA) inhibited the proliferation of human cervical cancer cell lines C33A, HeLa and CaSki, the reverse effect being observed in ORP4L overexpressing cells. For molecular insight, we found that ORP4L maintained intracellular Ca2+ homeostasis. Through this mechanism, ORP4L activated nuclear factor of activated T cells (NFAT) activity and thus promoted expression of a gene cluster which supported cell proliferation. Of note, ORP4L sustained inositol-1,4,5-trisphosphate receptor 1 (IP3R1) expression at both mRNA and protein levels via Ca2+-dependent NFAT3 activation, which offered a mechanic explanation for the role of ORP4L intracellular Ca2+ homeostasis. Furthermore, ORP4L knockdown markedly inhibited tumor growth in a C33A cell xenograft mouse model. To conclude, our results reveal that ORP4L promotes cell proliferation through maintaining intracellular Ca2+ homeostasis.
Assuntos
Biomarcadores Tumorais/metabolismo , Cálcio/metabolismo , Proliferação de Células , Homeostase/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Esteroides/metabolismo , Neoplasias do Colo do Útero/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Citoplasma/metabolismo , Feminino , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/metabolismo , Oxisteróis/metabolismo , Isoformas de Proteínas , Receptores de Esteroides/genética , Células Tumorais Cultivadas , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Using the oxidative modification of low density lipoprotein (LDL) as a model and the content of thiobabituric acid reactive substances (TBARS) s an index, the inhibitory effects of quercetin and rutin on oxidative modification of LDL induced by Fe(2+) and Cu(2+) were compared. The results showed that the rates of oxidative modification of LDL induced by Fe(2+) and Cu(2+) were similar in 0.15 mM NaCl (pH 7.4). The inhibitory action of quercetin on the oxidative modification of LDL induced by Cu(2+) was slightly more effective than in the case of induction by Fe(2+), and the action of rutin on the oxidation modification induced by Fe(2+) was significantly more effective than in the case of induction by Cu(2+).