RESUMO
A family of 14-20kDa, disulfide-rich, calcium-dependent secreted phospholipases A2 (sPLA2s) that release fatty acids from the sn-2 position of glycerophospholipids can be found in mammals. They have a diverse array of tissue distribution and biological functions. In this chapter we provide detailed protocols for production of nearly all of the mouse and human sPLA2s mainly by expression in bacteria and in vitro refolding or by expression in insect cells. High-resolution mass spectrometry and enzymatic assays were, respectively, used to show that all disulfides are formed and that the enzymes are active, strongly suggesting that each sPLA2 was prepared in the structurally native form. The availability of these proteins has allowed kinetic studies to be carried out, to prepare highly selective antisera, to screen for selective inhibitors, to study receptor binding, and to study the action of each enzyme on mammalian cell membranes and their in vivo biological roles.
Assuntos
Expressão Gênica , Fosfolipases A2 Secretórias/metabolismo , Fosfolipídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Animais , Baculoviridae/genética , Baculoviridae/metabolismo , Clonagem Molecular , Dissulfetos/química , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Fator Xa/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Células HEK293 , Humanos , Hidrólise , Corpos de Inclusão/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Camundongos , Fosfolipases A2 Secretórias/química , Fosfolipases A2 Secretórias/genética , Fosfolipídeos/química , Redobramento de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Células Sf9 , Spodoptera , Distribuição TecidualRESUMO
The M-type phospholipase A2 receptor (PLA2R1) is a member of the C-type lectin superfamily and can internalize secreted phospholipase A2 (sPLA2) via endocytosis in non-cancer cells. sPLA2 itself was recently shown to be overexpressed in prostate tumors and to be a possible mediator of metastasis; however, little is known about the expression of PLA2R1 or its function in prostate cancers. Thus, we examined PLA2R1 expression in primary prostate cells (PCS-440-010) and human prostate cancer cells (LNCaP, DU-145, and PC-3), and we determined the effect of PLA2R1 knockdown on cytotoxicity induced by free or liposome-encapsulated chemotherapeutics. Immunoblot analysis demonstrated that the expression of PLA2R1 was higher in prostate cancer cells compared to that in primary prostate cells. Knockdown of PLA2R1 expression in PC-3 cells using shRNA increased cell proliferation and did not affect the toxicity of cisplatin, doxorubicin (Dox), and docetaxel. In contrast, PLA2R1 knockdown increased the in vitro toxicity of Dox encapsulated in sPLA2 responsive liposomes (SPRL) and correlated with increased Dox and SPRL uptake. Knockdown of PLA2R1 also increased the expression of Group IIA and X sPLA2. These data show the novel findings that PLA2R1 is expressed in prostate cancer cells, that PLA2R1 expression alters cell proliferation, and that PLA2R1 modulates the behavior of liposome-based nanoparticles. Furthermore, these studies suggest that PLA2R1 may represent a novel molecular target for controlling tumor growth or modulating delivery of lipid-based nanomedicines.
