RESUMO
Protein glutathionylation is an important post-translational modification that regulates many cellular processes, including energy metabolism, signal transduction, and protein homeostasis. Global profiling of glutathionylated proteins (denoted as glutathionylome) is crucial for understanding redox-regulated signal transduction. Here, we developed a novel method based on click reaction and proteomics to enrich and identify the glutathionylated peptides in Escherichia coli and Drosophila lysates, in which 937 and 1,930 potential glutathionylated peptides were identified, respectively. Bioinformatics analysis showed that the cysteine residue next to negatively charged amino acid residues has a higher frequency of glutathionylation. Importantly, we found that most proteins associated with metabolic pathways were glutathionylated and that the glutathionylation sites of metabolic enzymes were highly conserved among different species. Our results indicate that the glutathione analog is a useful tool to characterize protein glutathionylation, and glutathionylation of metabolic enzymes, which play important roles in regulating cellular metabolism, is conserved.
Assuntos
Proteínas de Drosophila/química , Drosophila/metabolismo , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Glutationa/análogos & derivados , Sondas Moleculares/química , Sequência de Aminoácidos , Animais , Ciclo do Ácido Cítrico , Química Click , Creatina Quinase Forma MM/química , Creatina Quinase Forma MM/genética , Creatina Quinase Forma MM/metabolismo , Cisteína/química , Cisteína/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Escherichia coli/metabolismo , Glutationa/síntese química , Humanos , Malato Desidrogenase/antagonistas & inibidores , Malato Desidrogenase/metabolismo , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/química , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Proteômica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Adaptor protein ClpS is an essential regulator of prokaryotic ATP-dependent protease ClpAP, which delivers certain protein substrates with specific amino acid sequences to ClpAP for degradation. However, ClpS also functions as the inhibitor of the ClpAP-mediated protein degradation for other proteins. Here, we constructed the clpS-overexpression Mycobacterium smegmatis strain, and showed for the first time that overexpression of ClpS increased the resistance of M. smegmatis to rifampicin that is one of most widely used antibiotic drugs in treatment of tuberculosis. Using quantitative proteomic technology, we systematically analyzed effects of ClpS overexpression on changes in M. smegmatis proteome, and proposed that the increased rifampicin resistance was caused by ClpS-regulated drug sedimentation and drug metabolism. Our results indicate that the changes in degradation related proteins enhanced drug resistance and quantitative proteomic analysis is an important tool for understanding molecular mechanisms responsible for bacteria drug resistance.
Assuntos
Farmacorresistência Bacteriana , Endopeptidase Clp/metabolismo , Mycobacterium smegmatis/metabolismo , Proteômica , Rifampina/farmacologia , Proteases Dependentes de ATP/metabolismo , Mycobacterium smegmatis/efeitos dos fármacos , ProteóliseRESUMO
The identification of specific substrates of glutathione S-transferases (GSTs) is important for understanding drug metabolism. A method termed bioorthogonal identification of GST substrates (BIGS) was developed, in which a reduced glutathione (GSH) analogue was developed for recognition by a rationally engineered GST to label the substrates of the corresponding native GST. A K44G-W40A-R41A mutant (GST-KWR) of the mu-class glutathione S-transferases GSTM1 was shown to be active with a clickable GSH analogue (GSH-R1) as the cosubstrate. The GSH-R1 conjugation products can react with an azido-based biotin probe for ready enrichment and MS identification. Proof-of-principle studies were carried to detect the products of GSH-R1 conjugation to 1-chloro-2,4-dinitrobenzene (CDNB) and dopamine quinone. The BIGS technology was then used to identify GSTM1 substrates in the Chinese herbal medicine Ganmaocongji.
Assuntos
Glutationa Transferase/metabolismo , Química Click , Medicamentos de Ervas Chinesas/química , Medicamentos de Ervas Chinesas/metabolismo , Glutationa Transferase/química , Glutationa Transferase/genética , Espectrometria de Massas , Modelos Moleculares , Análise Serial de Proteínas , Engenharia de Proteínas , Especificidade por SubstratoRESUMO
The Fc-III tag is a newly developed fusion tag that can be applied to protein purification and detection. In the present work, we use the Fc-III-tagged green fluorescent protein (GFP) and human muscle creatine kinase (CK) as model systems to investigate effects of the Fc-III tag on activities and stabilities of the expressed multicysteine-containing proteins. Our results show the Fc-III tag has no adverse effects on the fluorescence of GFP and reduces the occurrence of GFP misfolding due to incorrect Cys oxidation compared with the His-tagged protein. The activity and stability of the Fc-III-tagged CK is slightly lower than that of the tag-free CK, but is higher than that of the His-tagged CK as determined by the ratio of the oxidized versus reduced CK. A major portion of His-tagged CK is in its oxidized form, while that of the Fc-III-tagged CK is in its reduced form. A folding model of CK with different tags was proposed, which may provide insights into the effect of the Fc-III tag on the conformations of disulfide-bridged proteins.