Trends in Biochemical Sciences
ReviewProtein quality control: U-box-containing E3 ubiquitin ligases join the fold
Section snippets
QC systems
The major sites of protein synthesis are the cytosol and the endoplasmic reticulum (ER), and multiple QC mechanisms are employed in these locations 1., 11.. The ER is the portal of entry for proteins into the secretory pathway. Glycoprotein QC is critical for monitoring the folding of proteins within the ER 11., 12.. As glycoproteins fold in the ER, specific sugars within the attached oligosaccharide chains are trimmed by glucosidases that function in conjunction with a network of
Pathways for conjugation of proteins with ubiquitin
Targeting a protein to the proteasome for degradation requires the selective addition of a polyubiquitin chain to the ill-fated polypeptide. Protein ubiquitination is a multistep process that involves at least three classes of enzymes (Fig. 1) 9., 10.. Enzymes of the E1 class utilize ATP hydrolysis to form a thioester-linked complex between glycine at the C-terminal of the 76-residue ubiquitin and cysteine within the E1 active site. Activated ubiquitin is then transferred from the E1 enzyme to
HECT and RING domain E3 enzymes
Several mechanistically distinct families of E3 enzymes facilitate the selection of proteins for ubiquitination. The HECT and the RING domain E3 enzymes were the first to be identified. HECT-domain E3 enzymes represent a family of ∼20 human proteins that select and transfer ubiquitin to substrates [10]. All HECT-domain E3 enzymes contain a 350-residue C-terminal domain that contains an active site cysteine that accepts ubiquitin from an E2 enzyme [23] and divergent regions in the N-terminus of
RING domain E3 enzymes in QC
Recently, five proteins that contain cytosolic RING E3 domains and function in QC have been identified (Table 2). These RING domain E3 enzymes act in a cullin-independent manner and include the yeast proteins Hrd1/Der3, Tul1 and Doa10, and the mammalian proteins Gp78 and Parkin 24., 25., 26., 27., 28.. Apart from Tul1, all are active in ERAD. They are either transmembrane ER proteins (Hrd1/Der3, Doa10 and Gp78) or cytosolic (Parkin) and function with the ER-membrane-localized E2 enzymes Ubc6
U-box proteins as non-canonical RING domain E3 proteins
Recent studies indicate that U-box-containing proteins form a third class of proteins that function as E3 enzymes. The U-box is a 75-amino-acid domain that was first identified in UFD2, the yeast E4 enzyme [35]. UFD2 is one of two U-box proteins in yeast, and in humans at least six family members fall into five subclasses (Table 3). Sequence analysis of the Arabidopsis genome predicts that plants have 37 U-box proteins [36]. The Koonin group has published BLAST analyses, sequence alignments and
Functions for U-box family members in QC
To enable the formation of complexes with chaperones, UFD2, CHIP and Cyc4 have either a polypeptide binding domain fused to the U-box domain or a protein–protein interaction domain (Table 3). Thus, these U-box proteins have the potential to facilitate the ubiquitination of chaperone substrates and may thereby facilitate aspects of QC and stress protection. Cyc4 is localized to the nucleus, and it is predicted that the cyclophilin domain of Cyc4 functions as a chaperone that has peptidyl-prolyl
How CHIP functions as a chaperone-dependent E3
How CHIP promotes chaperone-dependent ubiquitination of chaperones substrates is not clear. However, CHIP does not appear to inhibit the ability of Hsp40 to load proteins onto Hsp70, because it does not decrease the concentration of Hsp70–substrate complexes [21]. In addition, the combined action of Hsp70 and Hsp40 are required for CHIP to ubiquitinate denatured luciferase [40]. Thus, CHIP appears to function after substrate has bound to Hsp70.
Association with Hsp90 abrogates the interaction
Delivery of ubiquitinated, chaperone-bound substrates to the proteasome
How a chaperone-bound, ubiquitinated polypeptide is delivered to the proteasome for degradation is another unanswered question. It is possible that polyubiquitinated, non-native proteins are released from chaperones directly into the cytosol before recognition by the proteasome. However, this seems unlikely because polyubiquitinated proteins are prone to aggregation. Alternatively, CHIP, Hsp70 and/or Hsp90 may associate with other cofactors that facilitate the delivery of ubiquitinated
Regulation of chaperone function in protein folding and degradation
In current models of QC, the kinetics of folding controls the partitioning of non-native proteins between chaperones and proteases [1]. However, the identification of CHIP as a co-chaperone that converts Hsp70 and Hsp90 into multisubunit E3 enzyme complexes infers that the balance between folding and degradation of non-native proteins is regulated. For instance, 2–4-fold elevation of CHIP is sufficient to divert folding intermediates of the CFTR and GR from their normal biogenic pathways to the
Acknowledgements
The NIH and Cystic Fibrosis Foundation fund Work in the laboratory of DMC. Deutsche Forschungsgemeinschaft funds JH. The NIH supports the work of CP. Because of space concerns we were unable to include original citations to all of the literature reviewed.
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