Ubiquitin modifications of target proteins act to localise, direct and specify a diverse range of cellular processes, many of which are biomedically relevant. To allow this diversity, ubiquitin modifications exhibit remarkable complexity, determined by a combination of polyubiquitin chain length, linkage type, numbers of ubiquitin chains per target, and decoration of ubiquitin with other small modifiers. However, many questions remain about how different ubiquitin signals are specifically recognised and transduced by the decoding ubiquitin-binding domains (UBDs) within ubiquitin-binding proteins. This review briefly outlines our current knowledge surrounding the diversity of UBDs, identifies key challenges in their discovery and considers recent structural studies with implications for the increasing complexity of UBD function and identification. Given the comparatively low numbers of functionally characterised polyubiquitin-selective UBDs relative to the ever-expanding variety of polyubiquitin modifications, it is possible that many UBDs have been overlooked, in part due to limitations of current approaches used to predict their presence within the proteome. Potential experimental approaches for UBD discovery are considered; web-based informatic analyses, Next-Generation Phage Display, deubiquitinase-resistant diubiquitin, proximity-dependent biotinylation and Ubiquitin-Phototrap, including possible advantages and limitations. The concepts discussed here work towards identifying new UBDs which may represent the ‘dark matter’ of the ubiquitin system.
Functional analyses of PDB (Paget's disease of bone)-associated mutants of the p62 [also known as SQSTM1 (sequestosome 1)] signalling adaptor protein represent an interesting paradigm for understanding not only the disease mechanism in this skeletal disorder, but also the critical determinants of ubiquitin recognition by an ubiquitin-binding protein. The 11 separate PDB mutations identified to date all affect the C-terminal region of p62 containing the UBA domain (ubiquitin-associated domain), a ubiquitin-binding element. All of these mutations have deleterious effects on ubiquitin binding by p62 in vitro , and there is evidence of an inverse relationship between ubiquitin-binding function and disease severity. The effects on ubiquitin-binding function of most of the mutations can be attributed to either reduced UBA domain stability, and/or the mutations affecting the presumed ubiquitin-binding interface of the UBA domain. However, a subset of the mutations are more difficult to rationalize; several of these affect sequences of p62 outside of the minimal ubiquitin-binding region, providing insights into non-UBA domain sequences within the host protein which mediate ubiquitin-binding affinity. The p62 mutations are presumed to result in activation of (osteoclast) NF-κB (nuclear factor κB) signalling. Understanding how loss of ubiquitin-binding function of p62 impacts on signal transduction events in osteoclasts will undoubtedly further our understanding of the disease mechanism in PDB at the molecular level.
Mutations affecting the UBA (ubiquitin-associated) domain of SQSTM1 (Sequestosome 1) (p62) are a common cause of Paget's disease of bone. The missense mutations resolve into those which retain [P392L (Pro 392 →Leu), G411S] or abolish (M404V, G425R) the ability of the isolated UBA domain to bind Lys-48-linked polyubiquitin. These effects can be rationalized with reference to the solution structure of the UBA domain, which we have determined by NMR spectroscopy. The UBA domain forms a characteristic compact three-helix bundle, with a hydrophobic patch equivalent to that previously implicated in ubiquitin binding by other UBA domains. None of the mutations affect overall folding of the UBA domain, but both M404V and G425R involve residues in the hydrophobic patch, whereas Pro-392 and Gly-411 are more remote. A simple model assuming the isolated UBA domain is functioning as a compact monomer can explain the effects of the mutations on polyubiquitin binding. The P392L and G411S mutations do however have subtle local effects on secondary structure, which may become more relevant in full-length SQSTM1. Identification of the in vivo ubiquitylated substrates of SQSTM1 will be most informative in determining the functional significance of the SQSTM1–ubiquitin interaction, and consequences of the disease-associated mutations.