• 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • Given the important regulatory roles


    Given the important regulatory roles Ub signaling pathways play in eukaryotes, bacterial pathogens and viruses have evolved elaborate mechanisms to hijack host Ub signaling pathways and evade the immune response. This is highlighted by the numerous E3 Ub ligases and spectrum of DUBs encoded by bacterial and viral human pathogens (Maculins et al., 2016, Sheedlo et al., 2015). Since prokaryotic systems are essentially devoid of Ub, it is assumed that all bacterial effectors of Ub are present solely to manipulate the host. Therefore, in many instances bacteria simply employ their own E3 Ub ligases (generally a HECT-type) while relying on the native E1 and E2 enzymes of the host (Maculins et al., 2016). However, in a stunning turn, Qiu et al. (2016) characterize SdeA, a novel E3 Ub ligase of Legionella pneumophila capable of transferring Ub to Rab substrates independent of E1 and E2 enzymes. Of the ∼300 CB-5083 synthesis proteins L. pneumophila secretes within host cells, many are employed to override substrate selectivity of host SCF E3 complexes (Bruckert and Abu Kwaik, 2016, Quaile et al., 2015). Well-documented examples include LubX, a U-Box containing protein, and F-box proteins AnkB and LegU1. Interestingly, DUB activity in the N-terminal domain of SdeA has been previously characterized, as well as for two other SidE effector family members SdeB and SdeC (Sheedlo et al., 2015). Although K63-linked polyUb is the preferred linkage type for SdeA1–193, the structure of the domain is not related to any of the five DUB families (Figure 1B), but rather the ULP1 fold, characteristic of SUMO-specific proteases (Sheedlo et al., 2015). From sequence analysis downstream of the DUB domain, Qiu et al. (2016) discovered a canonical R-S-ExE motif within SdeA, constituting a putative mono-ADP-ribosyltransferase (mART) domain (Figure 1B). As demonstrated on yeast and a protozoan host, the R-S-ExE motif is essential for virulence and manipulation of the ER. Notably, two point mutations (R-S-AxA) abolished virulence and intracellular function of L. pneumophila. However, widespread ADP-ribosylation activity was not detected using 32P-NAD in human lysates. Yet, the mART motif was required to induce a molecular weight shift in ER-associated Rab proteins. Surprisingly, mass spectrometry analysis of “modified” Rab33b revealed the presence of Ub. This finding directed Qiu et al. (2016) to reconstitute an assay to identify the responsible E2 enzyme, but Rab33b was ubiquitinated under many unexpected conditions. Notably, ubiquitination still proceeded without ATP or Mg+2 and also following heat deactivation of lysate, which contains E1 and E2 enzymes. Even addition of the SdeA DUB domain and maleimide treatment did not alter the ubiquitinating activity of SdeA, highlighting a unique mechanism. In line with the ADP-ribosylating function of the mART motif, ubiquitination was found to be mainly dependent on the presence of nicotinamide adenine dinucleotide (NAD). This left the gaping question as to how Ub transfer by SdeA was related to NAD. In a most unconventional fashion, SdeA was still able to perform ubiquitination using Ub lacking the two C-terminal glycine residues and all possible Ub lysine mutants. However, modification of Rab33b appeared dependent on R42 of Ub, while also producing free nicotinamide. This suggested that SdeA was still functioning as a canonical mono-ADP-ribosyltransferase. Indeed, careful mass spectrometry analysis confirmed ADP-ribose was transferred to R42 of Ub. This finding was the missing link as to how Ub transfer was dependent on NAD. In a two-step process, ADP-ribose is added to R42 of Ub, which is then transferred to Rab substrates (Figure 1C). Qiu et al. (2016) demonstrate significant autoubiquitination of SdeA178–1,100 but can direct Ub transfer only to substrates using a truncated form, SdeA519–1,100. From a mechanistic view, this suggests that transfer of mono-ADP-ribosylated-Ub proceeds directly to the substrate. Future studies will be required to determine the mechanism of SdeA-catalyzed Ub transfer and how Ub is attached to the substrates.