Sjaak van Voorden

TEB4 is a C4HC3 RING finger-containing ubiquitin ligase of the endoplasmic reticulum

In the present study, the human TEB4 is identified as a novel ER (endoplasmic reticulum)-resident ubiquitin ligase. TEB4 has homologues in many species and has a number of remarkable properties. TEB4 contains a conserved RING (really interesting new gene) finger and 13 predicted transmembrane domains. The RING finger of TEB4 and its homologues is situated at the N-terminus and has the unconventional C4HC3 configuration. The N-terminus of TEB4 is located in the cytosol. We show that the isolated TEB4 RING domain catalyses ubiquitin ligation in vitro in a reaction that is ubiquitin Lys48-specific and involves UBC7 (ubiquitin-conjugating enzyme 7). These properties are reminiscent of E3 enzymes, which are involved in ER-associated protein degradation. TEB4 is an ER degradation substrate itself, promoting its own degradation in a RING finger- and proteasome-dependent manner.

The E3 Ubiquitin Ligases Hrd1 and gp78 Bind to and Promote Cholera Toxin Retro-Translocation

Cholera toxin intoxicates cells by trafficking from the cell surface to the endoplasmic reticulum (ER) where the toxic CTA1 peptide subsequently retro-translocates to the cytosol to induce toxicity. In this study, we uncovered the ER membrane components Hrd1 and gp78 as crucial players in the ER-to-cytosol transport of CTA1.

Ubiquitination of MHC Class I Heavy Chains Is Essential for Dislocation by Human Cytomegalovirus-encoded US2 but Not US11

The human cytomegalovirus-encoded glycoproteins US2 and US11 target newly synthesized major histocompatibility complex class I heavy chains for degradation by mediating their dislocation from the endoplasmic reticulum back into the cytosol, where they are degraded by proteasomes. A functional ubiquitin system is required for US2- and US11-dependent dislocation of the class I heavy chains. It has been assumed that the class I heavy chain itself is ubiquitinated during the dislocation reaction. To test this hypothesis, all lysines within the class I heavy chain were substituted. The lysine-less class I molecules could no longer be dislocated by US2 despite the fact that the interaction between the two proteins was maintained. Interestingly, US11 was still capable of dislocating the lysine-less heavy chains into the cytosol. Ubiquitination does not necessarily require lysine residues but can also occur at the N terminus of a protein. To investigate the potential role of N-terminal ubiquitination in heavy chain dislocation, a lysine-less ubiquitin moiety was fused to the N terminus of the class I molecule. This lysine-less fusion protein was still dislocated in the presence of US11. Ubiquitination could not be detected in vitro, either for the lysine-less heavy chains or for the lysine-less ubiquitin-heavy chain fusion protein. Our data show that although dislocation of the lysineless class I heavy chains requires a functional ubiquitin system, the heavy chain itself does not serve as the ubiquitin acceptor. This finding sheds new light on the role of the ubiquitin system in the dislocation process.

Novel Salmonella enterica Serovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication

ABSTRACT Upon contact with host cells, the intracellular pathogenSalmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribedSalmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonellachromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104 to 107bacteria in C3H/HeN and 101 to 104 bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.