RESUMO
Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP2) and regulate Ca2+ and protein kinase signaling in virtually all mammalian cell types. Chronic activation of the PLCϵ isoform downstream of G protein-coupled receptors (GPCRs) contributes to the development of cardiac hypertrophy. We have previously shown that PLCϵ-catalyzed hydrolysis of Golgi-associated phosphatidylinositol 4-phosphate (PI4P) in cardiac myocytes depends on G protein ßγ subunits released upon stimulation with endothelin-1. PLCϵ binds and is directly activated by Ras family small GTPases, but whether they directly interact with Gßγ has not been demonstrated. To identify PLCϵ domains that interact with Gßγ, here we designed various single substitutions and truncations of WT PLCϵ and tested them for activation by Gßγ in transfected COS-7 cells. Deletion of only a single domain in PLCϵ was not sufficient to completely block its activation by Gßγ, but blocked activation by Ras. Simultaneous deletion of the C-terminal RA2 domain and the N-terminal CDC25 and cysteine-rich domains completely abrogated PLCϵ activation by Gßγ, but activation by the GTPase Rho was retained. In vitro reconstitution experiments further revealed that purified Gßγ directly interacts with a purified fragment of PLCϵ (PLCϵ-PH-RA2) and increases PIP2 hydrolysis. Deletion of the RA2 domain decreased Gßγ binding and eliminated Gßγ stimulation of PIP2 hydrolysis. These results provide first evidence that Gßγ directly interacts with PLCϵ and yield insights into the mechanism by which ßγ subunits activate PLCϵ.
Assuntos
Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Complexo de Golgi/enzimologia , Miócitos Cardíacos/enzimologia , Fosfoinositídeo Fosfolipase C/metabolismo , Fosfatases cdc25/metabolismo , Animais , Células COS , Chlorocebus aethiops , Endotelina-1/genética , Endotelina-1/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Complexo de Golgi/genética , Miócitos Cardíacos/citologia , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoinositídeo Fosfolipase C/genética , Domínios Proteicos , Ratos , Fosfatases cdc25/genética , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
BACKGROUND AND PURPOSE: Cardiac ischaemia-reperfusion (IR) injury remains a significant clinical problem with limited treatment options available. We previously showed that cardioprotection against IR injury by nitro-fatty acids, such as nitro-linoleate (LNO2 ), involves covalent modification of mitochondrial adenine nucleotide translocase 1 (ANT1). Thus, it was hypothesized that conjugation of LNO2 to the mitochondriotropic triphenylphosphonium (TPP(+) ) moiety would enhance its protective properties. EXPERIMENTAL APPROACH: TPP(+) -LNO2 was synthesized from aminopropyl-TPP(+) and LNO2 , and characterized by direct infusion MS/MS. Its effects were assayed in primary cultures of cardiomyocytes from adult C57BL/6 mice and in mitochondria from these cells, exposed to simulated IR (SIR) conditions (oxygen and metabolite deprivation for 1h followed by normal conditions for 1h) by measuring viability by LDH release and exclusion of Trypan blue. Nitro-alkylated mitochondrial proteins were also measured by Western blots, using antibodies to TPP(+) . KEY RESULTS: TPP(+) -LNO2 protected cardiomyocytes from SIR injury more potently than the parent compound LNO2 . In addition, TPP(+) -LNO2 modified mitochondrial proteins, including ANT1, in a manner sensitive to the mitochondrial uncoupler carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and the ANT1 inhibitor carboxyatractyloside. Similar protein nitro-alkylation was obtained in cells and in isolated mitochondria, indicating the cell membrane was not a significant barrier to TPP(+) -LNO2 . CONCLUSIONS AND IMPLICATIONS: Together, these results emphasize the importance of ANT1 as a target for the protective effects of LNO2 , and suggest that TPP(+) -conjugated electrophilic lipid compounds may yield novel tools for the investigation of cardioprotection.