Abstract
The p62 protein (also known as SQSTM1) mediates diverse cellular functions including control of NFkappaB signaling and transcriptional activation. p62 binds non-covalently to ubiquitin and co-localizes with ubiquitylated inclusions in a number of human protein aggregation diseases. Mutations in the gene encoding p62 cause Paget's disease of bone (PDB), a common disorder of the elderly characterized by excessive bone resorption and formation. All of the p62 PDB mutations identified to date cluster within the C-terminal region of the protein, which shows low sequence identity to previously characterized ubiquitin-associated (UBA) domains. We report the first NMR structure of a recombinant polypeptide that contains the C-terminal UBA domain of the human p62 protein (residues 387-436). This sequence, which confers multiubiquitin chain binding, forms a compact three-helix bundle with a structure analogous to the UBA domains of HHR23A but with differences in the loop regions connecting helices that may be involved in binding accessory proteins. We show that the Pro392 --> Leu PDB substitution mutation modifies the structure of the UBA domain by extending the N terminus of helix 1. In contrast to the p62 PDB deletion mutations that remove the UBA domain and ablate multiubiquitin chain binding, the Pro392 --> Leu substitution does not affect interaction of the UBA domain with multiubiquitin chains. Thus, phenotypically identical substitution and deletion mutations do not appear to predispose to PDB through a mechanism dependent on a common loss of ubiquitin chain binding by p62.
Highlights
The ubiquitin-binding protein p62 (SQSTM1) functions as a scaffold in a range of signalling pathways associated with cell stress, survival, and inflammation, and controls transcriptional activation and protein recruitment to endosomes [1]. p62 co-localises with ubiquitin-containing inclusions in the brain tissue of Alzheimer’s, Pick’s and Parkinson’s disease patients, suggesting a possible role in the formation of these lesions [2], and recently, mutations in the gene encoding p62 have been found to cause Paget’s disease of bone (PDB) [3, 4], a common disorder of the elderly characterised by excessive bone resorption and formation [5]
We report the first structure of a recombinant polypeptide containing the UBA domain of the human p62 protein and NMR investigations of the Pro392Leu mutant
Structure calculations based on 735 NOE and 56 dihedral angle restraints show that the structure is well defined between residues Pro392 and Ile431, the short N- and C-terminal regions are much more dynamic and show no signs of stable secondary structure (Figure 2)
Summary
The ubiquitin-binding protein p62 (SQSTM1) functions as a scaffold in a range of signalling pathways associated with cell stress, survival, and inflammation, and controls transcriptional activation and protein recruitment to endosomes [1]. p62 co-localises with ubiquitin-containing inclusions in the brain tissue of Alzheimer’s, Pick’s and Parkinson’s disease patients, suggesting a possible role in the formation of these lesions [2], and recently, mutations in the gene encoding p62 have been found to cause Paget’s disease of bone (PDB) [3, 4], a common disorder of the elderly characterised by excessive bone resorption and formation [5]. All of the p62 mutations identified to date (Pro392Leu, Glu396Stop, and an intron 7 splice donor site mutation predicted to generate a frameshift 390Stop protein) cluster within the C-terminal region of the protein (Figure 1a). This region shows low sequence identity to previously characterised ubiquitin-associated (UBA) domains [6, 7] which occur in enzymes of the ubiquitin conjugation/deconjugation pathway [6], as well as in proteins that are downstream regulators of ubiquitin-dependent proteolysis [8]. This structural model allows us to rationalise the differential effects on ubiquitin-binding of p62 PDB deletion and substitution mutations detected in parallel in vitro ubiquitin chain-binding assays
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call