Phosphoribosylation of Ubiquitin Promotes Serine Ubiquitination and Impairs Conventional Ubiquitination

Abstract

Conventional ubiquitination involves the ATP-dependent formation of amide bonds between the ubiquitin C terminus and primary amines in substrate proteins. Recently, SdeA, an effector protein of pathogenic Legionella pneumophila, was shown to mediate NAD-dependent and ATP-independent ubiquitin transfer to host proteins. Here, we identify a phosphodiesterase domain in SdeA that efficiently catalyzes phosphoribosylation of ubiquitin on a specific arginine via an ADP-ribose-ubiquitin intermediate. SdeA also catalyzes a chemically and structurally distinct type of substrate ubiquitination by conjugating phosphoribosylated ubiquitin to serine residues of protein substrates via a phosphodiester bond. Furthermore, phosphoribosylation of ubiquitin prevents activation of E1 and E2 enzymes of the conventional ubiquitination cascade, thereby impairing numerous cellular processes including mitophagy, TNF signaling, and proteasomal degradation. We propose that phosphoribosylation of ubiquitin potently modulates ubiquitin functions in mammalian cells.

Deciphering the catalytic mechanism of bacterial ubiquitination

“…This revealed that the Arg42 residue in ubiquitin that becomes modified is located too far away from the ribose group of NADH for modification to occur directly . By contrast, another of ubiquitin's arginine residues, Arg72, which was previously shown to be important in SdeA-mediated ubiquitination 7 , is located much closer to the enzyme-bound NADH. The authors used computer simulations of the complex, called molecular dynamics, to show that Arg72 and one other arginine residue (Arg74) anchor ubiquitin to mART.…”

“…Isolating a sufficient number of nanoribbons that have perfect zigzag edges to enable their magnetic characterization is extremely challenging, and so the data from such studies 6 are scarce and inconclusive. Experiments performed on single graphene layers prepared in situ under a high vacuum have revealed the formation of local electronic states at edges, but did not provide any evidence of magnetism 7 . By expanding a previously developed…”

“…The discovery 6 of the SidE family of ubiquitin ligase enzymes in the bacterial pathogen Legionella pneumophila revealed a ubiquitination pathway with striking differences from the eukaryotic system. Not only can SidE ligases carry out the complete process without the aid of other enzymes, but this pathway also generates a different form of ubiquitin, termed phosphoribosylated ubiquitin (PR-Ub), in which a phosphoribose-sugar linkage attaches ubiquitin to the target protein 7 .…”

“…In the first step, the mono-ADP-ribosyltransferase (mART) domain of SdeA uses ubiquitin and NAD + to covalently attach an adenosine diphosphate ribose (ADPR) molecule to an arginine residue (Arg42) of ubiquitin 6 , producing an ADPRUb molecule. The phospho diesterase (PDE) domain of SdeA then cleaves this ADPR-Ub to release the molecule AMP, generating PR-Ub, and this group forms a bond with a serine residue in the target protein 7 . However, the molecular details of how ubiquitination is catalysed were a mystery until now.…”

“…If a water molecule enters the PDE domain's active site instead of a serine amino-acid residue, the reaction product released is unbound PR-Ub. PR-Ub can inhibit host E1-dependent ubiquitination because the PR modification prevents this form of ubiquitin from being a substrate for eukaryotic ubiquitination enzymes 7 . Kalayil et al answered the question of whether the pathogenicity associated with SdeA arises from the generation of unbound PR-Ub or from the ubiquitination of host proteins.…”