RESUMO
Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, "KAPRK," where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.
Assuntos
Arginina/metabolismo , Ácido Elágico/metabolismo , Histonas/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Ácido Elágico/química , Regulação da Expressão Gênica , Histonas/química , Histonas/genética , Humanos , Lythraceae/química , Metilação , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Prolina/metabolismo , Estrutura Terciária de Proteína , Proteína-Arginina N-Metiltransferases/química , Proteína-Arginina N-Metiltransferases/genética , Termodinâmica , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Xenopus laevisRESUMO
Single-chain Fv (scFv) format protein is a commonly used analytical tool for diagnostic and therapeutic applications. The usage of scFv antibody fragments in therapeutic applications has demonstrated that they need to have high thermostability. Many rational or irrational methods have been described erstwhile to engineer or improve the stability of scFv proteins by modifications of natural amino acid. Here we have demonstrated an alternate strategy to efficiently improve the thermostability of scFvs by non-canonical amino acid. Previously, fluoroprolines have been proven as a choice to tune the stability of many polypeptides and few globular proteins. Hence we exploited the usage of fluoroproline to enhance the thermal stability of scFv by replacing the natural proline on the framework regions of scFv that influence the folding or stability. To demonstrate our approach, a bacterial cytoplasmic foldable and humanized anti-c-Met scFv (hu-MscFv) was used. The hu-MscFv proline sites were successfully incorporated with (2S,4R)-4-fluoroproline without affecting its structure and function by the in vivo residue specific global replacement method which exploits bacterial auxotrophic system. The time-dependent temperature effect on the activity of fluorinated hu-MscFv revealed its increased stability at 40 °C along with improved half-life than the hu-MscFv with natural proline. Further model based structure analysis on hu-MscFv with fluoroproline indicated that the fluorine atoms were able to establish new favourable dipole interactions with neighbouring polar groups in their local micro environments that rationalizes its improved thermostability. Moreover the scFv sequence based statistical analysis strongly supports the fact that this method can be applied to any target scFv, since they contain high frequency conserved proline sites in their framework regions.