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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 lysine-less 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. [ABSTRACT FROM AUTHOR]

Abstract: 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 β2-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 β2m-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. [Copyright &y& Elsevier]

Growth hormone receptor (GHR) endocytosis is a highly regulated process that depends on the binding and activity of the multimeric ubiquitin ligase, SCFβTrCP (Skp Cullin F-box). Despite a specific interaction between β-transducin repeat-containing protein (βTrCP) and the GHR, and a strict requirement for ubiquitination activity, the receptor is not an obligatory target for SCFβTrCP-directed Lys48 polyubiquitination. We now show that also Lys63-linked ubiquitin chain formation is required for GHR endocytosis. We identified both the ubiquitin- conjugating enzyme Ubc13 and the ubiquitin ligase COOH terminus of Hsp70 interacting protein (CHIP) as being connected to this process. Ubc13 activity and its interaction with CHIP precede endocytosis of GHR. In addition toβTrCP, CHIP interacts specifically with the cytosolic tails of the dimeric GHR, identifying both Ubc13 and CHIP as novel factors in the regulation of cell surface availability of GHR. [ABSTRACT FROM AUTHOR]

A proper cellular adaptation to low oxygen levels is essential for processes such as development, growth, metabolism, and angiogenesis. The response to decrease in oxygen supply, referred to as hypoxia, is also involved in numerous human diseases including cancer, inflammatory conditions, and vascular disease. The hypoxia-inducible factor 1-α (HIF-1α), a key player in the hypoxic response, is kept under stringent regulation. At normoxia, the levels are kept low as a consequence of the efficient degradation by the ubiquitin-proteasome system, and in response to hypoxia, the degradation is blocked and the accumulating HIF-1α promotes a transcriptional response essential for proper adaptation and survival. Here we show that the ubiquitin-specific protease-19 (USP19) interacts with components of the hypoxia pathway including HIF-1α and rescues it from degradation independent of its catalytic activity. In the absence of USP19, cells fail to mount an appropriate response to hypoxia, indicating an important role for this enzyme in normal or pathological conditions. [ABSTRACT FROM AUTHOR]

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 nonglycosylated, 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[sup 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-α and CD3-δ. [ABSTRACT FROM AUTHOR]