Human cytomegalovirus-encoded US2 and US11 target unassembled MHC class I heavy chains for degradation.

Surface MHC class I molecules serve important immune functions as ligands for both T and NK cell receptors for the elimination of infected and malignant cells. In order to reach the cell surface, MHC class I molecules have to fold properly and form trimers consisting of a heavy chain (HC), a beta2-microglobulin light chain and an 8-10-mer peptide. A panel of ER chaperones facilitates the folding and assembly process. Incorrectly assembled or folded MHC class I HCs are detected by the ER quality-control system and transported to the cytosol for degradation by proteasomes. In human cytomegalovirus-infected cells, two viral proteins are synthesized, US2 and US11, which target MHC class I HCs for proteasomal degradation. It is unknown at which stage of MHC class I folding and complex formation US2 and US11 come into play. In addition, it is unclear if the disposal takes place via the same pathway through which proteins are removed that fail to pass ER quality control. In this study, we show with a beta2m-deficient cell line that US2 and US11 both target unassembled HCs for degradation. This suggests that US2 and US11 both act at an early stage of MHC class I complex formation. In addition, our data indicate that US11-mediated degradation involves mechanisms that are similar to those normally used to remove terminally misfolded HCs.

The ubiquitin-domain protein HERP forms a complex with components of the endoplasmic reticulum associated degradation pathway.

To eliminate misfolded proteins that accumulate in the endoplasmic reticulum (ER) the cell mainly relies on ubiquitin-proteasome dependent ER-associated protein degradation (ERAD). Proteolysis of ERAD substrates by the proteasome requires their ubiquitylation and retro-translocation from the ER to the cytoplasm. Here we describe a high molecular mass protein complex associated with the ER membrane, which facilitates ERAD. It contains the ubiquitin domain protein (UDP) HERP, the ubiquitin protein ligase HRD1, as well as the retro-translocation factors p97, Derlin-1 and VIMP. Our data on the structural arrangement of these ERAD proteins suggest that p97 interacts directly with membrane-resident components of the complex including Derlin-1 and HRD1, while HERP binds directly to HRD1. We propose that ubiquitylation, as well as retro-translocation of proteins from the ER are performed by this modular protein complex, which permits the close coordination of these consecutive steps within ERAD.

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.

To cause disease, cholera toxin (CT) is transported from the cell surface to the endoplasmic reticulum (ER) lumen where the catalytic CTA1 subunit retro-translocates to the cytosol to induce pathological water secretion. Two retro-translocon components are the Derlins and ER-associated multi-spanning E3 ubiquitin ligases including Hrd1 and gp78. We demonstrated previously that Derlin-1 facilitates CTA1 retro-translocation. However, as CTA1 is neither ubiquitinated on lysines nor at its N-terminus, the role of E3 ligases in toxin retro-translocation is unclear. Here, we show that expression of mutant Hrd1 and gp78 and a mutant E2-conjugating enzyme dedicated to retro-translocation (Ube2g2) decrease CTA1 retro-translocation. Hrd1 knockdown also attenuated toxin retro-translocation. Binding studies demonstrate that Hrd1 and gp78 interact with CT and protein disulfide isomerase, an ER chaperone that unfolds CTA1 to initiate translocation. Moreover, we find that the toxin's association with Hrd1 and gp78 is blocked by dominant-negative Derlin-1, suggesting that CT is targeted initially to Derlin-1 and then transferred to Hrd1 and gp78. These data demonstrate a role of the E3 ubiquitin ligases in CTA1 retro-translocation, implicate a sequence of events experienced by the toxin on the ER membrane, and raise the possibility that ubiquitination is involved in the transport process.

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.

Recruitment of the p97 ATPase and ubiquitin ligases to the site of retrotranslocation at the endoplasmic reticulum membrane.

Misfolded proteins are eliminated from the endoplasmic reticulum (ER) by retrotranslocation into the cytosol, a pathway hijacked by certain viruses to destroy MHC class I heavy chains. The translocation of polypeptides across the ER membrane requires their polyubiquitination and subsequent extraction from the membrane by the p97 ATPase [also called valosin-containing protein (VCP) or, in yeast, Cdc48]. In higher eukaryotes, p97 is bound to the ER membrane by a membrane protein complex containing Derlin-1 and VCP-interacting membrane protein (VIMP). How the ubiquitination machinery is recruited to the p97/Derlin/VIMP complex is unclear. Here, we report that p97 interacts directly with several ubiquitin ligases and facilitates their recruitment to Derlin-1. During retrotranslocation, a substrate first interacts with Derlin-1 before p97 and other factors join the complex. These data, together with the fact that Derlin-1 is a multispanning membrane protein forming homo-oligomers, support the idea that Derlin-1 is part of a retrotranslocation channel that is associated with both the polyubiquitination and p97-ATPase machineries.

IL-10 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin.

To determine to what extent lipopolysaccharide-induced IL-10 production capacity is determined by polymorphisms in toll-like receptor-4 (TLR4) and the IL-10 promoter region, we measured in vivo IL-10 and TNF-alpha production in patients undergoing elective cardiopulmonary bypass surgery, a major surgical trauma associated with ischemia-reperfusion injury that triggers an endotoxemia and profound inflammatory response in most patients. Ex vivo the IL-10 and TNF-alpha production was measured in a whole blood stimulation assay, using 3 LPS concentrations. Positive correlations were found between TNF-alpha and IL-10 production ex vivo, upon stimulation with each of the LPS concentrations. Also, the estimated TNF-alpha and IL-10 EC50, and TNF-alpha(max) and IL-10max were positively correlated (r = 0.203; p = 0.023 and r = 0.287; p = 0.001, respectively), indicating that these parameters describing LPS sensitivity and maximal production capacity, respectively, can be estimated by measuring either TNF-alpha or IL-10. Interleukin-10 concentrations in patients experiencing endotoxemia in vivo negatively correlated with the IL-10 levels produced upon stimulation with 1000 ng/mL LPS as well as the estimated IL-10max ex vivo. In vivo, a positive correlation between the TNF-alpha concentration at time-point 2 and the IL-10 concentration at time-point 3 was found, consistent with an important contribution of the magnitude of TNF-alpha release upon the subsequent IL-10 production. Carriers of the IL-10 promoter -1330G, -1082A, -819T, -592A (GATA) haplotype had lower IL-10 production ex vivo upon stimulation with 10 and 100 ng/mL LPS and higher EC50 values (the estimated LPS concentration at which 50% of the maximal IL-10 response is reached) as compared to carriers of the other haplotypes combined, indicating decreased LPS sensitivity ex vivo. These individuals did not differ from the others in interleukin-10 production capacity upon stimulation with a high LPS concentration (i.e., 1000 ng/mL) and the estimated IL-10(max) values, were similar, indicating unimpaired maximal IL-10 production capacity ex vivo. Carriers of the IL-10 promoter AGCC haplotype had lower EC50 values as compared to carriers of the other haplotypes combined, indicating increased LPS sensitivity ex vivo. In accordance with this finding, carriers of the AGCC haplotype had higher circulating IL-10 levels in vivo. The common TLR4 polymorphisms (Asp299Gly and Thr399Ile) were associated with slightly higher IL-10 production capacity ex vivo and in vivo, however, this was not statistically significant. Our results indicate that polymorphisms in the proximal IL-10 promoter region are associated with in vivo and ex vivo LPS sensitivity. The contribution to the inter-individual variation, however, is limited since the variation between individuals in LPS sensitivity and IL-10 production capacity can only partly be attributed to these IL-10 promoter polymorphisms.

Human HRD1 is an E3 ubiquitin ligase involved in degradation of proteins from the endoplasmic reticulum.

The ubiquitin system plays an important role in endoplasmic reticulum (ER)-associated degradation of proteins that are misfolded, that fail to associate with their oligomerization partners, or whose levels are metabolically regulated. E3 ubiquitin ligases are key enzymes in the ubiquitination process as they recognize the substrate and facilitate coupling of multiple ubiquitin units to the protein that is to be degraded. The Saccharomyces cerevisiae ER-resident E3 ligase Hrd1p/Der3p functions in the metabolically regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and additionally facilitates the degradation of a number of misfolded proteins from the ER. In this study we characterized the structure and function of the putative human orthologue of yeast Hrd1p/Der3p, designated human HRD1. We show that human HRD1 is a non-glycosylated, stable ER protein with a cytosolic RING-H2 finger domain. In the presence of the ubiquitin-conjugating enzyme UBC7, the RING-H2 finger has in vitro ubiquitination activity for Lys(48)-specific polyubiquitin linkage, suggesting that human HRD1 is an E3 ubiquitin ligase involved in protein degradation. Human HRD1 appears to be involved in the basal degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase but not in the degradation that is regulated by sterols. Additionally we show that human HRD1 is involved in the elimination of two model ER-associated degradation substrates, TCR-alpha and CD3-delta.

TNF-alpha promoter, Nod2 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin.

Humans exhibit substantial inter-individual differences in TNF-alpha production upon endotoxin stimulation. To determine to what extent the lipopolysaccharide-induced TNF-alpha production capacity in vivo and ex vivo is determined by polymorphisms in toll-like receptor-4 (TLR4), the TNF-alpha promoter region and Nod2, we screened for two TLR4 polymorphisms, a Nod2 polymorphism and the TNF-alpha promoter polymorphisms. We measured the perioperative endotoxemia and TNF-alpha production and the TNF-alpha production capacity of each patient in a whole-blood stimulation assay using blood drawn before anesthesia, using various LPS concentrations, in patients undergoing elective cardiac surgery. This operation represents a major surgical trauma associated with ischemia-reperfusion injury and triggers an endotoxemia and profound inflammatory response. In vivo TNF-alpha production was positively correlated with the level of endotoxemia after aortic declamping; thus TNF-alpha levels were higher in patients having endotoxemia compared to patients without endotoxemia. This correlation was observed in patients with any of the genotypes studied, and did not differ between the various genotypes. In vivo TNF-alpha levels correlated best with those ex vivo after stimulation with 1000 ng/mL LPS, and the estimated maximal TNF-alpha release capacity. Subjects with the wild-type TLR4 gene had similar levels of TNF-alpha upon LPS stimulation ex vivo as compared with patients carrying Asp299Gly and/or the Thr399Ile TLR4 polymorphism. Our results indicate that polymorphisms in the TLR4 receptor, Nod2 and TNF-alpha promoter region are not strongly associated with in vivo and ex vivo TNF-alpha production capacity upon endotoxin stimulation. This suggests that in this model of natural LPS release, the variation between individuals in TNF-alpha release can only modestly be determined by genetic background (TNF-alpha promoter, Nod2 and TLR4) of the individual.

Severe Mycobacterium bovis BCG infections in a large series of novel IL-12 receptor beta1 deficient patients and evidence for the existence of partial IL-12 receptor beta1 deficiency.

Cell mediated immunity plays a critical role in human host defence against intracellular bacteria. In patients with unusual, severe infections caused by poorly pathogenic species of mycobacteria and salmonellae, genetic deficiencies have been identified in key genes in the type-1 cytokine pathway, especially in IFNGR1 and IL12RB1. Here, we analyzed 11 patients originating from Turkey and suffering from unusual Mycobacterium bovis Bacille Calmette-Guerin infections following vaccination, and found that most patients (n=8) are deficient in IL-12Rbeta1 expression and function. No defects were found in patients' IFN-gammaR or IL-18R. In addition, a first patient suffering from partial IL-12Rbeta1 deficiency is described. This patient presented with an intermediate cellular and immunological phenotype: a consistent, low response to IL-12 was found, which could be further augmented by IL-18. Despite a lack of cell surface IL-12Rbeta1 expression, normal levels of intracellular IL-12Rbeta1 protein were detectable, which was not seen in the other, completely IL-12Rbeta1 deficient patients examined. Moreover, this patient had a relatively mild clinical phenotype and was the only individual with a single homozygous amino acid substitution in IL-12Rbeta1 (C198R). Collectively, our findings indicate that idiopathic, unusually severe infections due to M. bovis BCG can be caused by complete as well as partial IL-12Rbeta1 deficiency.