Identification Of Inhibitors Of Mycobacterium Tuberculosis Ubiquitin-like Protein Ligase Pafa And Pafa Interactome

Tuberculosis(TB)is among the most prevalent infectious diseases in the world in large part owing to the wide-spread emergence of multidrug resistance(MDR)and extensive drug resistance(XDR)and poor Mycobacterium tuberculosis(Mtb)basic research.There is thus a significant pressing need for the identification of potential drug targets and the underlying mechanism of TB pathogenesis to facilitate drug development.As an essential protein and the sole Prokaryotic Ubiquitinlike Protein(Pup)ligase of the Pup-Proteasome System(PPS)in Mtb,PafA is a very attractive drug target because there are no PafA homologs in humans and the PPS does not exist in many other bacteria.However,there are presently no effective inhibitors or known inhibitor-related mechanisms of PafA.And as a key component of the intracellular proteolytic system,PafA must be carefully regulated to prevent excessive protein degradation.Currently,those factors underlying PafA regulation remain poorly understood.Thus,in this thesis,we anticipate to identify an inhibitor-related mechanisms of Mtb PafA and based on this finding,with the computer-aided drug screening,to find 1-2 novel lead inhibitors for PafA.Meanwhile,based on our lab's Mtb proteome microarray,we aim to carry out the identification of PafA interactome and discover regulation mechanism of PafA.Firstly,we find that the pupylase activity of Mtb PafA are significantly inhibited upon the association of AEBSF(4-(2-aminoethyl)benzenesulfonyl fluoride)to Serine 119(S119).Mutation of S119 to amino acids that resemble AEBSF has similar inhibitory effects on PafA activity as that of AEBSF.Detailed structural analysis reveals that S119 is located at a critical position in the groove where PafA binds the C-terminal region of Pup though distant from the PafA catalytic site.Phenotypic studies demonstrate that PafA S119 is essential for Msm survival under nitrogen limitation and in macrophages.PafA S119-based inhibitor screening studies show inhibitor PHD-01-58 inhibit the pupylase activity of PafA significantly and render Mtb susceptible to nitirc oxide-mediated stress.On the other hand,we have applied the Mtb proteome microarray to systematic identify PafA interactome and identified 72 PafA binding proteins.With bioinformatic analysis,we analyzed the functional classification of the candidate protein.Further,in order to check on the reliability of the obtained PafA interactome,we used the BLI interaction technique to verify the affinity of candidate protein and PafA.Then through performing the Pupylation assays in vitro,we found five out of seven proteins which were validated by BLI technology can be pupylated catalyzed by pafA.Based on PafA interactome,we propose that Rv0998,as an acetyl transferase,may have a regulating effect on the activity of PafA combined with acetylated modification sites of PafA reported in previous literatures.Mutation of acetylated modification sites to glutamine that resemble acetylation has inhibitory effects on PafA activity.Phenotypic studies demonstrate that PafA K202 and K361 is essential for mycobacteria survival.Detailed structural analysis reveals that K202 and K361 is located at a critical position near the substrates binding domain and the pup binding domain respectively.Overall,in this study,we identify S119 as situated within a critical,small-molecule accessible region of PafA whose modification inhibits PafA activity and is thus a highly promising target for the development of PafA-specific inhibitor.Meanwhile,we provide PafA interactome based on the Mtb proteome microarray and acetylated regulation mechanism for PafA,which may serve as leads for in-depth functional and mechanistic study of Mtb pathogenesis involved in Pupylation.