Phosphoproteomics Reveals the AMPK Substrate Network in Response to DNA Damage and Histone Acetylation

AMP-activated protein kinase(AMPK)is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various substrates.Emerging studies have demonstrated the regulatory roles of AMPK in DNA repair,but the underlying mechanisms remain to be fully understood.Herein,using mass spectrometry-based proteomic technologies,we systematically investigate the regulatory network of AMPK in DNA damage response(DDR).Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biological processes,whereas our system-wide histone modification analysis reveals a link between AMPK and histone acetylation.Together with these findings,we discover that AMPK promotes apoptosis by phosphorylating apoptosis-stimulating of p53 protein 2(ASPP2)in an irradiation(IR)-dependent manner and regulates histone acetylation by phosphorylating histone deacetylase 9(HDAC9)in an IR-independent manner.Besides,we reveal that disrupting the histone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhances the sensitivity of AMPKdeficient cells to IR.Therefore,our study has provided a resource to investigate the interplay between phosphorylation and histone acetylation underlying the regulatory network of AMPK,which could be beneficial to understand the exact role of AMPK in DDR.

Nucleolus localization of SpyCas9 affects its stability and interferes with host protein translation in mammalian cells

The CRISPR/Cas9 system,originally derived from the prokaryotic adaptive immune system,has been developed as efficient genome editing tools.It enables precise gene manipulation on chromosomal DNA through the specific binding of programmable sgRNA to target DNA,and the Cas9 protein,which has endonuclease activity,will cut a double strand break at specific locus.However,Cas9 is a foreign protein in mammalian cells,and the potential risks associated with its introduction into mammalian cells are not fully understood.In this study,we performed pull-down and mass spectrometry(MS)analysis of Streptococcus pyogenes Cas9(SpyCas9)interacting proteins in HEK293T cells and showed that the majority of Cas9-associated proteins identified by MS were localized in the nucleolus.Interestingly,we further discovered that the Cas9 protein contains a sequence encoding a nucleolus detention signal(NoDS).Compared with wild-type(WT)Cas9,NoDS-mutated variants of Cas9(mCas9)are less stable,although their gene editing activity is minimally affected.Overexpression of WT Cas9,but not mCas9,causes general effects on transcription and protein translation in the host cell.Overall,identification of NoDS in Cas9 will improve the understanding of Cas9's biological function in vivo,and the removal of NoDS in Cas9 may enhance its safety for future clinical use.