RESUMO
Lysine succinylation is a newly identified protein post-translational modification pathway present in both prokaryotic and eukaryotic cells. However, succinylation substrates and regulatory enzyme(s) remain largely unknown, hindering the biological study of this modification. Here we report the identification of 2,580 bacterial lysine succinylation sites in 670 proteins and 2,803 lysine acetylation (Kac) sites in 782 proteins, representing the first lysine succinylation dataset and the largest Kac dataset in wild-type E. coli. We quantified dynamic changes of the lysine succinylation and Kac substrates in response to high glucose. Our data showed that high-glucose conditions led to more lysine-succinylated proteins and enhanced the abundance of succinyllysine peptides more significantly than Kac peptides, suggesting that glucose has a more profound effect on succinylation than on acetylation. We further identified CobB, a known Sir2-like bacterial lysine deacetylase, as the first prokaryotic desuccinylation enzyme. The identification of bacterial CobB as a bifunctional enzyme with lysine desuccinylation and deacetylation activities suggests that the eukaryotic Kac-regulatory enzymes may have enzymatic activities on various lysine acylations with very different structures. In addition, it is highly likely that lysine succinylation could have unique and more profound regulatory roles in cellular metabolism relative to lysine acetylation under some physiological conditions.
Assuntos
Acil Coenzima A/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Regulação Bacteriana da Expressão Gênica , Processamento de Proteína Pós-Traducional , Sirtuínas/metabolismo , Acetilação , Motivos de Aminoácidos , Cromatografia Líquida de Alta Pressão , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Glucose/metabolismo , Glucose/farmacologia , Marcação por Isótopo , Lisina/metabolismo , Anotação de Sequência Molecular , Mapeamento de Interação de Proteínas , Transdução de Sinais , Sirtuínas/genética , Especificidade por Substrato , Espectrometria de Massas em TandemRESUMO
Plasmodium falciparum Sir2A (PfSir2A), a member of the sirtuin family of nicotinamide adenine dinucleotide-dependent deacetylases, has been shown to regulate the expression of surface antigens to evade the detection by host immune surveillance. It is thought that PfSir2A achieves this by deacetylating histones. However, the deacetylase activity of PfSir2A is weak. Here we present enzymology and structural evidence supporting that PfSir2A catalyzes the hydrolysis of medium and long chain fatty acyl groups from lysine residues more efficiently. Furthermore, P. falciparum proteins are found to contain such fatty acyl lysine modifications that can be removed by purified PfSir2A in vitro. Together, the data suggest that the physiological function of PfSir2A in antigen variation may be achieved by removing medium and long chain fatty acyl groups from protein lysine residues. The robust activity of PfSir2A would also facilitate the development of PfSir2A inhibitors, which may have therapeutic value in malaria treatment.
Assuntos
Histonas/metabolismo , Lisina/metabolismo , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/metabolismo , Sirtuínas/metabolismo , Acilação , Sequência de Aminoácidos , Variação Antigênica/imunologia , Escherichia coli , Humanos , Hidrólise , Evasão da Resposta Imune , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/imunologia , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sirtuínas/química , Sirtuínas/genética , Especificidade por SubstratoRESUMO
To explore the impact of global incorporation of fluorinated aromatic amino acids on protein function, we investigated the effects of three monofluorinated phenylalanine analogs para-fluorophenylalanine (pFF), meta-fluorophenylalanine (mFF), and ortho-fluorophenylalanine (oFF) on the stability and enzymatic activity of the histone acetyltransferase (HAT), tGCN5. We selected this set of fluorinated amino acids because they bear the same size and overall polarity but alter in side chain shape and dipole direction. Our experiments showed that among three fluorinated amino acids, the global incorporation of pFF affords the smallest perturbation to the structure and function of tGCN5.
