Fluorescent Probes for Monitoring Serine Ubiquitination

Abstract

In a radical departure from the classical E1−E2−E3 three-enzyme mediated ubiquitination of eukaryotes, the recently described bacterial enzymes of the SidE family of Legionella pneumophila effectors utilize NAD + to ligate ubiquitin onto target substrate proteins. This outcome is achieved via a two-step mechanism involving (1) ADP ribosylation of ubiquitin followed by (2) phosphotransfer to a target serine residue. Here, using fluorescent NAD + analogues as well as synthetic substrate mimics, we have developed continuous assays enabling real-time monitoring of both steps of this mechanism. These assays are amenable to biochemical studies and high-throughput screening of inhibitors of these effectors, and the discovery and characterization of putative enzymes similar to members of the SidE family in other organisms. We also show their utility in studying enzymes that can reverse and inhibit this post-translational modification.

Arginine ADP-Ribosylation: Chemical Synthesis of Post-Translationally Modified Ubiquitin Proteins

“…Our next aim was to investigate the SdeA-mediated ligation of substrate ER-proteins to Ub ADPr , the critical biological process in the onset of Legionnaires' disease. 40 We synthesized a 20-mer peptide (sequence on page S29) derived from the ER remodeling RTN4b protein (23) known to be a substrate of SidE effectors, 8 equipped with a rhodamine fluorophore on the N-terminus. We tested whether SdeA, using its PDE domain, would ligate Ub ADPr to this RTN4b peptide to form a fluorescent peptide-Pr-Ub conjugate (Figure 4A).…”

Assay technologies facilitating drug discovery for ADP‐ribosyl writers, readers and erasers

“…[42] Multiple other fluorescent NAD + probes have been also recently reported [52,53] and applied to for example measuring NAD + dependent ubiquitination of Legionella pneumophila SidE proteins. [54] A method to label potential PARP substrate proteins in cells using a clickable NAD + was introduced by Jiang et al [34] When used in the reaction, clickable NAD + analogs provide terminal alkyne groups in the attached ADP-ribosylation, which can be subsequently conjugated with a suitable detectable affinity tag and visualized on SDS-PAGE gels. By using biotin as a label, isolation and identification of such substrate proteins is possible.…”

The unity of opposites: Strategic interplay between bacterial effectors to regulate cellular homeostasis

“…Several structures of different constructs of these proteins ( 126 , 127 , 128 , 129 ) ( Table 1 ), along with biochemical studies, have allowed elucidation of some critical aspects of the various catalytic steps involved in recognition of Ub by the mART domain and those involved in recognition of Ub-ADPR by the PDE domain. The initial discovery of the five substrates: Rtn4 (reticulon 4) Rab1a, Rab6a, Rab30, and Rab33b ( 29 , 122 ), was quickly followed by the understanding that the SidE proteins are tolerant of any serine that is a part of an unstructured/flexible region, provided it can be accommodated in the PDE active site ( 127 , 130 , 131 ). Since SidE proteins are known to colocalize with the LCV, it seems likely that the SidE enzymes target their substrates by proximity-based selection rather than by sequence specificity.…”