Lysine ¹¹ -Linked Polyubiquitination of the AnkB F-Box Effector of Legionella pneumophila

Abstract

The fate of the polyubiquitinated protein is determined by the lysine linkages involved in the polymerization of the ubiquitin monomers, which has seven lysine residues (K 6 , K 11 , K 27 , K 29 , K 33 , K 48 , and K 63 ). The translocated AnkB effector of the intravacuolar pathogen Legionella pneumophila is a bona fide F-box protein, which is localized to the cytosolic side of the Legionella-containing vacuole (LCV) and is essential for intravacuolar proliferation within macrophages and amoebae. The F-box domain of AnkB interacts with the host SCF1 E3 ubiquitin ligase that triggers the decoration of the LCV with K 48 -linked polyubiquitinated proteins that are targeted for proteasomal degradation. Here we report that AnkB becomes rapidly polyubiquitinated within the host cell, and this modification is independent of the F-box domain of AnkB, indicating host-mediated polyubiquitination. We show that the AnkB effector interacts specifically with the host E3 ubiquitin ligase Trim21. Mass spectrometry analyses have shown that AnkB is modified by K 11 -linked polyubiquitination, which has no effect on its stability. This work shows the first example of K 11 -linked polyubiquitination of a bacterial effector and its interaction with the host Trim21 ubiquitin ligase.

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

“…125 Another study showed that AnkB is also modified by Trim21-mediated K11-linked ubiquitination; however, this fails to change its stability. 126 LegU1 and LegAU13 are integrated into the host SCF E3 complex, with the former directly ubiquitinating BAT3, a host co-chaperone of Hsp70 that is implicated in ER stress modulation. 127 SidC and its paralog SdcA, identified in L. pneumophila, are capable of recruiting the ER-derived vesicles to build LCV.…”

Ubiquitination Accomplished: E1 and E2 Enzymes Were Not Necessary

“…Of the 300 effector 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.…”

The manipulation of host transcription by the ANKH effector of legionella.

“…HEK293T cells were transfected with 3XFLAG-AnkH, BAP, and c-myc-LARP7 for 24 h and collected in lysis buffer, as described previously [177,254]. FLAG-tagged and myc-tagged proteins were immunoprecipitated by using anti-FLAG M2 magnetic beads (Sigma) or SureBeads Protein G Magnetic Beads (BioRad) crosslinked with anti-myc antibody (ProteinTech).…”

“…Legionella pneumophila strains were lysed using B-PER (Thermo Scientific) and heated at 99°C for 5 minutes in sample buffer. 1 X 10 6 bacteria were loaded per lane and separated by 10.4 to 15% SDS-PAGE (BioRad), and transferred onto a polyvinylidene difluoride (PVDF) (BioRad) membrane, as described previously [254] Immunoprecipitated proteins were heated at 99°C for 5 minutes in sample buffer, separated by 10.4 to 15% SDS-PAGE (BioRad), and transferred onto a polyvinylidene difluoride (PVDF) (BioRad) membrane, as described previously [254]. Anti-Flag Biologics and was used at a dilution of 1:100 [186,187].Cya-hybrids were detected using monoclonal M-45 antibody at a dilution of 1:50 [53].…”