Global Protein Deacetylase Dicovery By "Clip-Chip" Techonolgy And A E.coli Proteome Microarray

Lysine acetylation is a reversible, dynamic, and highly conservedpost-translational modification (PTM) from prokaryotes to eukaryotes. Asan important post-translational modification, protein acetylation has beenextensively studied in the past few decades. Protein acetylationmodification related to many major fuctions (e.g. metabolism, cell cycle,apoptosis), abnormal protein acetylation level could cause some diseases,such as cancer, neurodegenerative disease, diabetes. Acetylation is alsorelated to gene transcription, pathways, and aging.Protein acetylation is a dynamic process, the existing research is apoint in time, or at most a few point-in-time snapshot detection. And due tothe limit of the sensitivity of mass spectrometry. Therefore, acetylation ofproteins has been found that a small part may only account for the actualexistence of acetylated proteins, even so, the number of acetylated proteinsis very large. But with a wide range of dynamic protein acetylation eventcorresponding to only a very small number of non-histone deacetylase. Forinstance, only CobB (Sir2homolog) was identified and studied in E.coli,In addition, CobB is not an essential gene of E.coli. In preliminaryexperiments, we also found that the knockout CobB does not affect theoverall level of acetylation of the E.coli Therefore, we speculate that thereare a certain number of protein deacetylase yet to be discovered.Researchers have developed numerous algorithms for functionprediction, and the series of BLAST algorithms is arguably mostsuccessful. However, such homology-based algorithms developed to dateare only capable of predicting divergent but not convergent functionality ofproteins, and as such perform rather poorly when the sequenceconservation is low, such as proteins in the Ser/Thr protease and protein acetyltransferase families.In this study, we develop a new method called “Clip-Chip” toidentify novel deacetylases based on an E.coli proteome chip (contains4,256proteins). Based on this approach, we identified a novel deacetylaseYcgC. Western blot resμLt shows that YcgC could deacetylates itssubstrate YcdC (RutR DNA-binding transcriptional dual regulator) In vitro.Furthermore, the HPLC MS/MS results indicate that the52and62acetylated lysine residue of YcdC could be deacetylated by YcgC.Moreover, we synthesized YcdC-Flag sequence and replaced the genomeYcdC by this tagged one, we purified the tagged YcdC and verified thatYcdC could also be deacetylated by YcgC In vivo.Moreover, we also measured the downstream gene alterations causedby YcdC acetyaltion level change, the real-time PCR results shows theGad system genes were down-regulated, indicates deacetylated YcdC hasstronger DNA binding activity.Above all, in this study, we proposed a new approach to identifynovel deactylases, and we validated the novel deacetylase which identifiedby “Clip-Chip” technology both In vitro and In vivo. Taken together, thismethod could be used in novel enzyme discovery (e.g. protease) in thefuture.