Ubiquitination independent of E1 and E2 enzymes by bacterial effectors

Abstract

Signaling by ubiquitination regulates virtually every cellular process in eukaryotes. Covalent attachment of ubiquitin to a substrate is catalyzed by the E1, E2 and E3 three-enzyme cascade 1, which links the C terminus of ubiquitin via an isopeptide bond mostly to the ε-amino group of a lysine of the substrate. Given the essential roles of ubiquitination in the regulation of the immune system, it is not surprising that the ubiquitination network is a common target for diverse infectious agents 2. For example, many bacterial pathogens exploit ubiquitin signaling using virulence factors that function as E3 ligases, deubiquitinases 3 or as enzymes that directly attack ubiquitin 4. The bacterial pathogen Legionella pneumophila utilizes approximately 300 effectors that modulate diverse host processes to create a niche permissive for its replication in phagocytes 5. Here we demonstrate that members of the SidE effector family (SidEs) of L. pneumophila ubiquitinate multiple Rab small GTPases associated with the endoplasmic reticulum (ER). Moreover, we show that these proteins are capable of catalyzing ubiquitination without the need for the E1 and E2 enzymes. A putative mono ADP-ribosyltransferase (mART) motif critical for the ubiquitination activity is also essential for the role of SidEs in intracellular bacterial replication in a protozoan host. The E1/E2-independent ubiquitination catalyzed by these enzymes is energized by NAD which activates ubiquitin by the formation of ADP-ribosylated ubiquitin (ADPR-Ub). These results establish that ubiquitination can be catalyzed by a single enzyme whose activity does not require ATP.

The ubiquitin system: a critical regulator of innate immunity and pathogen–host interactions

“…130 Finally, a peculiar yet interesting study recently reported that SdeA, belonging to the SidE family effectors of L. pneumophila, is a novel all-in-one ubiquitin conjugation enzyme, which solely mediates the ubiquitination of many host Rab GTPases by virtue of a putative mono-ADPribosyltransferase motif. 131 Of note, this report not only demonstrates for the first time that ubiquitination can be catalyzed by bacterial effectors in the absence of E1 and E2 enzymes but also hints that ubiquitin can be activated by ADP ribosylation.…”

Deciphering the catalytic mechanism of bacterial ubiquitination

“…The electronic and magnetic properties of graphene nanoribbons correlate closely with the structures of their edges, and are sensitive to even minute numbers of defects. 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 .…”

“…So far, only a few SdeA substrates have been identified 6,8,9 ; these include the GTPase enzymes Rab and Rag, as well as the protein RTN4. From analysis of the ubiquitination sites in host proteins, Kalayil et al and Wang et al propose that the ligase enzyme specifically targets serine residues in disordered protein regions.…”

“…The activated ubiquitin is then transferred to downstream enzymes, which attach ubiquitin through an isopeptide bond to a lysine amino-acid residue in the target protein. 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 .…”