RESUMO
Histone deacetylase 6 (HDAC6) is upregulated in a variety of tumor cell lines and has been linked to many cellular processes, such as cell signaling, protein degradation, cell survival, and cell motility. HDAC6 is an enzyme that deacetylates the acetyllysine residues of protein substrates, and the discovery of HDAC6 substrates, including tubulin, has revealed many roles of HDAC6 in cell biology. Unfortunately, among the wide variety of acetylated proteins in the cell, only a few are verified as HDAC6 substrates, which limits the full characterization of HDAC6 cellular functions. Substrate trapping mutants were recently established as a tool to discover unanticipated substrates of histone deacetylase 1 (HDAC1). In this study, we applied the trapping approach to identify potential HDAC6 substrates. Among the high confidence protein hits after trapping, protein arginine methyl transferase 5 (PRMT5) was successfully validated as a novel HDAC6 substrate. PRMT5 acetylation enhanced its methyltransferase activity and symmetrical dimethylation of downstream substrates, revealing possible crosstalk between acetylation and methylation. Substrate trapping represents a powerful, systematic, and unbiased approach to discover substrates of HDAC6.
Assuntos
Desacetilase 6 de Histona/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Acetilação , Domínio Catalítico/genética , DNA Helicases/metabolismo , Células HEK293 , Desacetilase 6 de Histona/química , Desacetilase 6 de Histona/genética , Humanos , Mutação , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteína-Arginina N-Metiltransferases/química , Proteômica/métodos , RNA Helicases/metabolismo , Proteínas com Motivo de Reconhecimento de RNA/metabolismoRESUMO
Histone deacetylaseâ 1 (HDAC1) regulates transcription by deacetylating histones. In addition to histones, several non-histone proteins are HDAC1 substrates, which suggests a role for HDAC1 beyond epigenetics. Unfortunately, the identification of non-histone substrates has been largely serendipitous, which makes full characterization of HDAC1 functions difficult. To overcome this challenge, inactive "trapping" mutants were recently developed to identify HDAC1 substrates. To optimize substrate trapping, the relative trapping abilities of 17 inactive HDAC1 mutants was assessed. HDAC1 H141A, F150A, and C151A showed strong binding to substrates LSD1 and p53. Interestingly, each mutant preferentially trapped a different substrate. By combining several inactive mutants, the trapping strategy will facilitate the discovery of new HDAC1 substrates and shed light on the variety of HDAC1-related functions in cell biology.
Assuntos
Histona Desacetilase 1/química , Histona Desmetilases/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Epigênese Genética , Células HEK293 , Histona Desacetilase 1/genética , Histonas/metabolismo , Humanos , Mutação , Ligação Proteica , Especificidade por SubstratoRESUMO
Histone deacetylase 1 (HDAC1) is an epigenetic enzyme that regulates key cellular processes, such as cell proliferation, apoptosis, and cell survival, by deacetylating histone substrates. Aberrant expression of HDAC1 is implicated in multiple diseases, including cancer. As a consequence, HDAC inhibitors have emerged as effective anti-cancer drugs. HDAC inhibitor-induced G0/G1 cell-cycle arrest has been attributed to epigenetic transcriptional changes mediated by histone acetylation. However, the mechanism of G2/M arrest remains poorly understood. Here, we identified mitosis-related protein Eg5 (KIF11) as an HDAC1 substrate using a trapping mutant strategy. HDAC1 colocalized with Eg5 during mitosis and influenced the ATPase activity of Eg5. Importantly, an HDAC1- and HDAC2-selective inhibitor caused mitotic arrest and monopolar spindle formation, consistent with a model in which Eg5 deacetylation by HDAC1 is critical for mitotic progression. These findings revealed a previously unknown mechanism of action of HDAC inhibitors involving Eg5 acetylation, and provide a compelling mechanistic hypothesis for HDAC inhibitor-mediated G2/M arrest.
