Identification And Mechanism Of An ADP-Ribosylarginine Hydrolase Reversing The Non-canonical Ubiquitination

Ubiquitination is one of the most important post-transcriptional modification that regulates various cellular processes in eukaryotes.Canonical ubiquitination is catalyzed by E1-E2-E3 cascade in an ATP-dependent manner.Recently,SidEs,an effector protein family of pathogenic Legionella pneumophila,was shown to mediate a non-canonical ubiquitination in which a phosphoribosyl ubiquitin(PR-Ub)is conjugated to substrates via a phosphodiester bond.Together with the non-canonical ubiquitination,ADPR-Ub and PR-Ub was processed and accumulated,which impairs the canonical Ub system.However,these functions of SidEs are spatially and temporally regulated,such as Dup A/Dup B and Sid J.Although these effectors make us understand the non-classical ubiquitination better,question still remains:is there any L.pneumophila effector which is able to reverse ADPR-Ub or PR-Ub to Ub?To answer this question,we prepared call lysate of L.pneumophila and found that the lysate could hydrolyze ADPR-Ub.With a screening system for the ADPRH (ADP-ribosyl hydrolase),We found that MavL(lpg2526)is the effector which hydrolyzed ADPR-Ub.Via biochemical methods and MALDI-TOF,we furtherly proved that ADPR-Ub was reversed to Ub by MavL.Additionally,MavL exhibited a high binding affinity to Ub and specifically hydrolyzes ADPR-Ub rather than ADPR-Actin,indicating that Ub is the substrate.Also,MavL could not hydrolyze ADPR^R66-Ub,suggesting that MavL is an ADP-ribosylarginine hydrolase.The lysate of Lp02?mav L could no longer hydrolyze ADPR-Ub.In conclusion,MavL is the unique ADP-ribosylarginine hydrolase specifically towards ADPR^R42-Ub.Our results showed that MavL(31-416aa)is a truncation with ADPRH activity.The E107 and D323 are key for catalysis and F105 is essential for Ub binding.MavL was anchored to endoplasmic reticulum and Golgi apparatus through its C-terminal.In 293T cells,MavL hydrolyzed ADPR-Ub generated by SidE and hijacked the conjugation of PR-Ub to substrates in cells.Also,MavL suppressed SdeA(m ART)toxicity in Saccharomyces cerevisiae.This study makes it possible for a better understanding of non-classical ubiquitination and AMP-ribosylation in mammal cells.