RESUMO
CGI-58/ABHD5 coactivates adipose triglyceride lipase (ATGL). In adipocytes, CGI-58 binds to perilipin 1A on lipid droplets under basal conditions, preventing interaction with ATGL. Upon activation of protein kinase A (PKA), perilipin 1A is phosphorylated and CGI-58 rapidly disperses into the cytoplasm, enabling lipase coactivation. Because the amino acid sequence of murine CGI-58 has a predicted PKA consensus sequence of RKYS(239)S(240), we hypothesized that phosphorylation of CGI-58 is involved in this process. We show that Ser239 of murine CGI-58 is a substrate for PKA using phosphoamino acid analysis, MS, and immuno-blotting approaches to study phosphorylation of recombinant CGI-58 and endogenous CGI-58 of adipose tissue. Phosphorylation of CGI-58 neither increased nor impaired coactivation of ATGL in vitro. Moreover, Ser239 was not required for CGI-58 function to increase triacylglycerol turnover in human neutral lipid storage disorder fibroblasts that lack endogenous CGI-58. Both CGI-58 and S239A/S240A-mutated CGI-58 localized to perilipin 1A-coated lipid droplets in cells. When PKA was activated, WT CGI-58 dispersed into the cytoplasm, whereas substantial S239A/S240A-mutated CGI-58 remained on lipid droplets. Perilipin phosphorylation also contributed to CGI-58 dispersion. PKA-mediated phosphorylation of CGI-58 is required for dispersion of CGI-58 from perilipin 1A-coated lipid droplets, thereby increasing CGI-58 availability for ATGL coactivation.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/química , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Espaço Intracelular/metabolismo , Serina/metabolismo , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Transporte/metabolismo , Chlorocebus aethiops , Colforsina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Espaço Intracelular/efeitos dos fármacos , Lipase/metabolismo , Masculino , Camundongos , Dados de Sequência Molecular , Perilipina-1 , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Mutations in the gene encoding comparative gene identification 58 (CGI-58)/α/ß hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. CGI-58 has been identified as a coactivator of adipose TG lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We developed a molecular model of CGI-58 structure and then mutated predicted active site residues and performed LPAAT activity assays of recombinant WT and mutated CGI-58. When mutations of predicted catalytic residues failed to reduce LPAAT activity, we determined that LPAAT activity was due to a bacterial contaminant of affinity purification procedures, plsC, the sole LPAAT in Escherichia coli Purification protocols were optimized to reduce plsC contamination, in turn reducing LPAAT activity. When CGI-58 was expressed in SM2-1(DE3) cells that lack plsC, lysates lacked LPAAT activity. Additionally, mouse CGI-58 expressed in bacteria as a glutathione-S-transferase fusion protein and human CGI-58 expressed in yeast lacked LPAAT activity. Previously reported lipid binding activity of CGI-58 was revisited using protein-lipid overlays. Recombinant CGI-58 failed to bind lysophosphatidic acid, but interestingly, bound phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 5-phosphate [PI(5)P]. Prebinding CGI-58 with PI(3)P or PI(5)P did not alter its coactivation of ATGL in vitro. In summary, purified recombinant CGI-58 that is functional as an ATGL coactivator lacks LPAAT activity.
Assuntos
1-Acilglicerol-3-Fosfato O-Aciltransferase/química , Aciltransferases/química , Lisofosfolipídeos/química , 1-Acilglicerol-3-Fosfato O-Aciltransferase/genética , 1-Acilglicerol-3-Fosfato O-Aciltransferase/metabolismo , Aciltransferases/genética , Aciltransferases/metabolismo , Animais , Humanos , Lisofosfolipídeos/genética , Lisofosfolipídeos/metabolismo , Camundongos , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Small cell lung cancer (SCLC) is a aggressive form of primary lung neoplasm that often presents in elderly smokers. While stage I SCLC can be managed with surgery, extensive-stage disease is managed with chemotherapy using etoposide and cisplatin among other agents, and often complemented by radiation therapy to the chest and cranium. Recent advances in pharmacological research have yielded novel antibody and peptide-conjugated adjunctive chemotherapy, of which bombesin and bombesin receptors have played an important role due to their overexpression in SCLC and other lung cancers. Chemotherapy agents conjugated to bombesin or bombesin-like peptides often demonstrate higher therapeutic efficacy, greater treatment specificity, as well as improved cytotoxicity towards SCLC cells that demonstrate drug resistance. Further modifications to the bombesin-drug conjugate, such as liposomal preparation, have further enhanced bio-availability and half-life of the compound. Additionally, bombesin-radioisotope conjugates can be used for early detection of SCLC using positron emission tomography, as well as subsequent targeted adjuvant radiotherapy to help minimize radiation-induced fibrosis of healthy tissue. Ultimately, further studies are imperative to capitalize on the various applications of bombesin conjugates in both the diagnosis and management of SCLC.