Tyler Florio’s Blog Spotlight – Ubiquitination independent of E1 and E2 enzymes by bacterial effectors
Ubiquitination is a vital cellular process universally observed in eukaryotes and is responsible for tagging protein substrates for various cellular functions such as degradation, modulation, transport, etc. This process is named after the 76 amino acid polypeptide called ubiquitin which is attached to designated protein substrates in three steps (activation, conjugation, and ligation respectively) via three specific enzymes (E1, E2, and E3 respectively). It has been demonstrated that the ubiquitination pathway is responsible for regulating vital cellular processes such as immune response.
Although bacteria does not utilize the ubiquitination pathway, many infectious bacteria species such as E. coli and Salmonella exploit ubiquitination’s role in immune response by developing toxic “ubiquitin-like” proteins called effectors. These effectors can function as either E3-ligases, deubiquitinases, and even attack ubiquitin attached to protein substrates which allows the bacteria to suppress the host’s immune system and allow it to survive. The classical method of ubiquitination involves the systematic cascade of E1, E2, and E3 to attach ubiquitin to its target; however, a new study by Qiu et al (doi: 10.1038/nature17657) demonstrated that the infectious bacteria L. pneumophila express an effector protein SdeA which performs a substrate ubiquitination independent of E1 and E2 enzymes.
The authors were originally investigating the SidE effector protein family (SdeA, SdeB, SdeC, and SdeD) for L. pneumophila which are the virulent effectors and discovered a conserved mono-ADP ribosyltransferase (mART) domain through protein-sequence analysis. Upon further investigation, the authors determined that mART domain in critical for SdeA-mediated toxicity, but surprisingly did not demonstrate ADP-ribosylation activity. The lack of ADP-ribosylation activity suggests that a different biochemical mechanism than previously thought was occurring
Perplexed, the authors performed a co-expression of WT and mART-mutated SdeA with ER-associated Rab proteins Rab1, Rab6A, Rab30, and Rab33b (known L. pneumophila target) and discovered Rab1 and Rab33b experienced a molecular mass shift with WT SdeA and not the mutant. Through mass spectrometry analysis, the authors determined that altered Rab1 and Rab33b had ubiquitin polypeptides attached to them while the unaltered Rab1 and Rab33b did not which suggests that SdeA performs a ubiquitination reaction with Rab1 and Rab33b. Then when the authors attempted an in vitro ubiquitination with WT SdeA and Rab33b in the presence of E1, ATP, and several E2’s and found Rab33b had not been ubiquitinated; however, the addition of cofactor NAD resulted in ubiquitination activity (Fig 1). The lack of ubiquitinated Rab33b protein with E1, ATP, and several E2’s suggests that the classical ubiquitination pathway does not apply to SdeA. This is the first reported case where a protein substrate is ubiquitinated independent of E1 and E2 dependence and suggests a currently unknown mechanism is occuring with SdeA which results in ubiquitination. This opens the door to determine if these effectors have other unknown substrates and if there exists other proteins which ubiquitinate independent of E1 and E2. Future investigations into these E1-E2 independent ubiquitinases with mART domains can expand our understanding of the cellular processes regulated by ubiquitination.
Reference: Qiu, Jiazhang, Michael J. Sheedlo, Kaiwen Yu, Yunhao Tan, Ernesto S. Nakayasu, Chittaranjan Das, Xiaoyun Liu, and Zhao-Qing Luo. “Ubiquitination Independent of E1 and E2 Enzymes by Bacterial Effectors.” Nature, 2016. doi:10.1038/nature17657.