RESUMO
Adipokinetic hormone (AKH) is the main hormone involved in the acute regulation of hemolymph lipid levels in several insects. In adult Manduca sexta AKH promotes a rapid phosphorylation of "Lipid storage protein-1", Lsd1, and a concomitant activation of the rate of hydrolysis of triglycerides by the main fat body lipase. In contrast, in the larval stage AKH modulates hemolymph trehalose levels. The present study describes the sequence of a full-length Lsd1 cDNA obtained from M. sexta fat body and investigates a possible link between Lsd1 expression and the distinct effects of AKH in larva and adult insects. The deduced protein sequence showed a high degree of conservation compared to other insect Lsd1s, particularly in the central region of the protein (amino acids 211-276) in which the predicted lipid binding helices are found. Lsd1 was absent in feeding larva and its abundance progressively increased as the insect develops from the non-feeding larva to adult. Contrasting with the levels of protein, Lsd1 transcripts were maximal during the feeding larval stages. The subcellular distribution of Lsd1 showed that the protein exclusively localizes in the lipid droplets. Lsd1 was found in the fat body but it was undetectable in lipid droplets isolated from oocytes or embryos. The present study suggests a link between AKH-stimulated lipolysis in the fat body and the expression of Lsd1.
Assuntos
Hormônios de Inseto/fisiologia , Proteínas de Insetos/metabolismo , Metabolismo dos Lipídeos , Manduca/metabolismo , Oligopeptídeos/fisiologia , Ácido Pirrolidonocarboxílico/análogos & derivados , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Conservada , DNA Complementar/química , Corpo Adiposo/metabolismo , Hormônios de Inseto/farmacologia , Proteínas de Insetos/análise , Proteínas de Insetos/genética , Manduca/efeitos dos fármacos , Manduca/genética , Manduca/crescimento & desenvolvimento , Dados de Sequência Molecular , Oligopeptídeos/farmacologia , Ácido Pirrolidonocarboxílico/farmacologia , Alinhamento de SequênciaRESUMO
The structure of Locusta migratoria apolipophorin-III consists of a five-helix bundle connected by four short loops. The role of the conformational flexibility of helices and loops on the lipid-binding activity of this apolipoprotein was investigated by disulfide mediated tethering experiments. One disulfide mutant tethering the second and fourth loops (L2-L4), and two disulfide mutants restricting the flexibility of the neighboring alpha-helices 3 and 4 (H3-H4) and 1 and 5 (H1-H5), were studied. The ability of the disulfide mutants to interact with phospholipid vesicles, mixed micelles of phosphatidylcholine and cholate, and in vivo with native spherical lipoprotein particles was studied. The L2-L4 mutant was active with native lipoproteins as well as being able to form discoidal lipoproteins upon incubation with either liposomes or discoidal micelles. The H3-H4 mutant was not able to interact with liposomes or native lipoproteins but interacted with discoidal micelles. The H1-H5 mutant was unable to interact with lipid in any of the three systems. Three conclusions were reached: (1) opening of the helix bundle does not require the separation of loops 2 and 4 as recently proposed by others and (2) alpha-helices 3 and/or 4 are involved in the insertion of apoLp-III in both phospholipid bilayers and monolayers. The conformational flexibility of helices 3 and 4 is required for the lipid-binding activity of apoLp-III. (3) Interaction of helices 1 and/or 5 with the lipid surface is required to the formation of stable lipoprotein complexes of any kind.
Assuntos
Apolipoproteínas/química , Proteínas de Insetos/química , Lipoproteínas/química , Animais , Apolipoproteínas/genética , Apolipoproteínas/metabolismo , Cisteína/química , Cisteína/genética , Dimiristoilfosfatidilcolina/química , Dimiristoilfosfatidilcolina/metabolismo , Dissulfetos/química , Gafanhotos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lipoproteínas/metabolismo , Manduca , Micelas , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Conformação Proteica , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , UltracentrifugaçãoRESUMO
An original method for the study of the lipid binding properties of exchangeable apolipoproteins is reported. Binding of Locusta migratoria apolipophorin-III to Manduca sexta low-density lipophorin (LDLp) and high-density lipophorin (HDLp) was studied in vivo. This assay could be used useful to investigate the effect of mutations in the lipid binding properties of exchangeable apolipoproteins under physiological conditions.