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1.
J Lipid Res ; 51(6): 1566-80, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20028662

RESUMO

Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identification methods. We first report that a synthetic omega-alkynyl-palmitate analog can be readily and specifically incorporated into GAPDH or mitochondrial 3-hydroxyl-3-methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fluorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or flat bed fluorescence scanning. The acylated cysteine residues were confirmed by MS. Second, omega-alkynyl-palmitate is preferentially incorporated into transiently expressed H- or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with omega-alkynyl-myristate or omega-alkynyl-stearate, via an alkali sensitive thioester bond. Third, omega-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of omega-alkynyl-palmitate and omega-alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with omega-alkynyl-palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H- and N-Ras could be easily detected using omega-alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The omega-alkynyl-myristate and -palmitate analogs used with click chemistry and azido-probes will be invaluable to study protein acylation in vitro, in cells, and in vivo.


Assuntos
Alcinos/química , Ácidos Graxos/química , Ácidos Graxos/metabolismo , Proteínas/química , Proteínas/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Enzimas/metabolismo , Humanos , Espaço Intracelular/metabolismo , Células Jurkat , Lipoilação , Camundongos , Dados de Sequência Molecular , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Fatores de Tempo , Proteínas ras/química , Proteínas ras/metabolismo
2.
FASEB J ; 22(3): 797-806, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17932026

RESUMO

Myristoylation is the attachment of the 14-carbon fatty acid myristate to the N-terminal glycine residue of proteins. Typically a co-translational modification, myristoylation of proapoptotic cysteinyl-aspartyl proteases (caspase)-cleaved Bid and PAK2 was also shown to occur post-translationally and is essential for their proper localization and proapoptotic function. Progress in the identification and characterization of myristoylated proteins has been impeded by the long exposure times required to monitor incorporation of radioactive myristate into proteins (typically 1-3 months). Consequently, we developed a nonradioactive detection methodology in which a bio-orthogonal azidomyristate analog is specifically incorporated co- or post-translationally into proteins at N-terminal glycines, chemoselectively ligated to tagged triarylphosphines and detected by Western blotting with short exposure times (seconds to minutes). This represents over a million-fold signal amplification in comparison to using radioactive labeling methods. Using rational prediction analysis to recognize putative internal myristoylation sites in caspase-cleaved proteins combined with our nonradioactive chemical detection method, we identify 5 new post-translationally myristoylatable proteins (PKC epsilon, CD-IC2, Bap31, MST3, and the catalytic subunit of glutamate cysteine ligase). We also demonstrate that 15 proteins undergo post-translational myristoylation in apoptotic Jurkat T cells. This suggests that post-translational myristoylation of caspase-cleaved proteins represents a novel mechanism widely used to regulate cell death.


Assuntos
Apoptose , Azidas/química , Ácidos Mirísticos/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Animais , Western Blotting , Células COS , Linhagem Celular , Técnicas de Química Analítica/métodos , Chlorocebus aethiops , Glicina/química , Glicina/metabolismo , Humanos , Células Jurkat , Ácidos Mirísticos/química , Proteínas/análise , Proteínas/química , Fatores de Tempo
3.
FASEB J ; 22(3): 721-32, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17971398

RESUMO

Increased levels of circulating saturated free fatty acids, such as palmitate, have been implicated in the etiology of type II diabetes and cancer. In addition to being a constituent of glycerolipids and a source of energy, palmitate also covalently attaches to numerous cellular proteins via a process named palmitoylation. Recognized for its roles in membrane tethering, cellular signaling, and protein trafficking, palmitoylation is also emerging as a potential regulator of metabolism. Indeed, we showed previously that the acylation of two mitochondrial proteins at their active site cysteine residues result in their inhibition. Herein, we sought to identify other palmitoylated proteins in mitochondria using a nonradioactive bio-orthogonal azido-palmitate analog that can be selectively derivatized with various tagged triarylphosphines. Our results show that, like palmitate, incorporation of azido-palmitate occurred on mitochondrial proteins via thioester bonds at sites that could be competed out by palmitoyl-CoA. Using this method, we identified 21 putative palmitoylated proteins in the rat liver mitochondrial matrix, a compartment not recognized for its content in palmitoylated proteins, and confirmed the palmitoylation of newly identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. We postulate that covalent modification and perhaps inhibition of various mitochondrial enzymes by palmitoyl-CoA could lead to the metabolic impairments found in obesity-related diseases.


Assuntos
Acil Coenzima A/química , Azidas/química , Ácidos Graxos/química , Lipoilação , Proteínas Mitocondriais/metabolismo , Ácido Palmítico/metabolismo , Acil Coenzima A/biossíntese , Animais , Azidas/metabolismo , Células Cultivadas , Ácidos Graxos/metabolismo , Hepatócitos/metabolismo , Humanos , Hidroximetilglutaril-CoA Sintase/metabolismo , Mitocôndrias Hepáticas/enzimologia , Proteínas Mitocondriais/química , Estrutura Molecular , Ácido Palmítico/química , Ratos
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