Tesis doctoral de Albert Gubern Burset
Lipid droplets (ld) are organelles present in all cell types, consisting of a hydrophobic core of triacylglycerols (tag) and cholesteryl esters, surrounded by a monolayer of phospholipids and cholesterol. The present work shows that ld biogenesis induced by serum, by long-chain fatty acids, or the combination of both in cho-k1 cells was prevented by phospholipase a2 inhibitors with a pharmacological profile consistent with the implication of group iva pla2 (cpla2). Knocking down cpla2 expression with sirna was similar to pharmacological inhibition in terms of enzyme activity and ld biogenesis. A cho cell clone stably expressing an enhanced green fluorescent protein-cpla2 fusion protein (egfp-cpla2) displayed higher ld occurrence under basal conditions and upon ld induction. Induction of ld took place with concurrent phosphorylation of cpla2 at ser505. Transfection of a s505a mutant cpla2 showed that phosphorylation at ser505 is key for enzyme activity and ld formation. Cpla2 contribution to ld biogenesis was not due to the generation of arachidonic acid, nor was related to neutral lipid synthesis. Cpla2 inhibition in cells induced to form ld resulted in the appearance of tubulo-vesicular profiles of the smooth er, compatible with a role of cpla2 in the formation of nascent ld from the er. Our results suggest that cpla2 is regulated independently of calcium and dependent of jnk pathway and ceramide kinase. Treatment of cho-k1 cells with inhibitors of fatty acid synthase and in the absence of external sources of fatty acids induced the synthesis of triacylglycerol (tag) from pre-existing fatty acids and also lipid droplet (ld) biogenesis. Tag synthesis was required for ld biogenesis and was sensitive to pharmacological inhibition and downregulation of the expression of group via phospholipase a2 (ipla2-via). Similar results were obtained after induction of stress with acidic ph, c2-ceramide, tunicamycin, or deprivation of glucose. In contrast, ld induction with lipoproteins present in serum did not involve tag synthesis from endogenous fatty acids and was not dependent on ipla2-via. Overexpression of the enzyme enhanced basal tag synthesis from endogenous fatty acids and ld occurrence, and increased the effects of stress inducers. During stress, ld biogenesis but not tag synthesis required phosphorylation and activation of group iva pla2 (cpla2). The results demonstrate that ipla2-via operates providing fatty acids for tag synthesis while cpla2 allows ld biogenesis. Ld biogenesis during stress may represent a pro-surviving strategy to recycle structural phospholipids into energy-generating substrates.
Datos académicos de la tesis doctoral «Role of phospholipases a2 (group iva and via) in the biogenesis of intracelular lipid dropiets«
- Título de la tesis: Role of phospholipases a2 (group iva and via) in the biogenesis of intracelular lipid dropiets
- Autor: Albert Gubern Burset
- Universidad: Autónoma de barcelona
- Fecha de lectura de la tesis: 30/09/2008
Dirección y tribunal
- Director de la tesis
- Enrique Claro Izaguirre
- Tribunal
- Presidente del tribunal: jose Rodriguez alvarez
- ramón Trullas oliva (vocal)
- suzanne Jackowski (vocal)
- joan-marc Servitja duque (vocal)