Jed Long

Ubiquitin Recognition by the Ubiquitin-associated Domain of p62 Involves a Novel Conformational Switch

The p62 protein functions as a scaffold in signaling pathways that lead to activation of NF-κB and is an important regulator of osteoclastogenesis. Mutations affecting the receptor activator of NF-κB signaling axis can result in human skeletal disorders, including those identified in the C-terminal ubiquitin-associated (UBA) domain of p62 in patients with Paget disease of bone. These observations suggest that the disease may involve a common mechanism related to alterations in the ubiquitin-binding properties of p62. The structural basis for ubiquitin recognition by the UBA domain of p62 has been investigated using NMR and reveals a novel binding mechanism involving a slow exchange structural reorganization of the UBA domain to a “bound” non-canonical UBA conformation that is not significantly populated in the absence of ubiquitin. The repacking of the three-helix bundle generates a binding surface localized around the conserved Xaa-Gly-Phe-Xaa loop that appears to optimize both hydrophobic and electrostatic surface complementarity with ubiquitin. NMR titration analysis shows that the p62-UBA binds to Lys48-linked di-ubiquitin with ∼4-fold lower affinity than to mono-ubiquitin, suggesting preferential binding of the p62-UBA to single ubiquitin units, consistent with the apparent in vivo preference of the p62 protein for Lys63-linked polyubiquitin chains (which adopt a more open and extended structure). The conformational switch observed on binding may represent a novel mechanism that underlies specificity in regulating signalinduced protein recognition events.

Loss of Ubiquitin Binding Is a Unifying Mechanism by Which Mutations of SQSTM1 Cause Paget’s Disease of Bone

Ubiquitin-associated (UBA) domain mutations of SQSTM1 are an important cause of Paget’s disease of bone (PDB), which is a human skeletal disorder characterized by abnormal bone turnover. We previously showed that, when introduced into the full-length SQSTM1 protein, the disease-causing P392L, M404V, G411S, and G425R missense mutations and the E396X truncating mutation (representative of all of the SQSTM1 truncating mutations) cause a generalized loss of monoubiquitin binding and impaired K48-linked polyubiquitin binding at physiological temperature. Here, we show that the remaining three known PDB missense mutations, P387L, S399P, and M404T, have similar deleterious effects on monoubiquitin binding and K48-linked polyubiquitin binding by SQSTM1. The P387L mutation affects an apparently unstructured region at the N terminus of the UBA domain, some five residues from the start of the first helix, which is dispensable for polyubiquitin binding by the isolated UBA domain. Our findings support the proposal that the disease mechanism in PDB with SQSTM1 mutations involves a common loss of ubiquitin binding function of SQSTM1 and implicate a sequence extrinsic to the compact globular region of the UBA domain as a critical determinant of ubiquitin recognition by the full-length SQSTM1 protein.

Dimerisation of the UBA Domain of p62 Inhibits Ubiquitin Binding and Regulates NF-κB Signalling

The ubiquitin (Ub)-binding p62 scaffold protein (encoded by the SQSTM1 gene) regulates a diverse range of signalling pathways leading to activation of the nuclear factor kappa B (NF-kappaB) family of transcription factors and is an important regulator of macroautophagy. Mutations within the gene encoding p62 are commonly found in patients with Pagets disease of bone and largely cluster within the C-terminal ubiquitin-associated (UBA) domain, impairing its ability to bind Ub, resulting in dysregulated NF-kappaB signalling. However, precisely how Ub-binding is regulated at the molecular level is unclear. NMR relaxation dispersion experiments, coupled with concentration-dependent NMR, CD, isothermal titration calorimetry and fluorescence kinetic measurements, reveal that the p62 UBA domain forms a highly stable dimer (K(dim) approximately 4-12 microM at 298 K). NMR analysis shows that the dimer interface partially occludes the Ub-binding surface, particularly at the C-terminus of helix 3, making UBA dimerisation and Ub-binding mutually exclusive processes. Somewhat unusually, the monomeric UBA appears to be the biologically active form and the dimer appears to be the inactive one. Engineered point mutations in loop 1 (E409K and G410K) are shown to destabilise the dimer interface, lead to a higher proportion of the bound monomer and, in NF-kappaB luciferase reporter assays, are associated with reduced NF-kappaB activity compared with wt-p62.

Characterization of a non-UBA domain missense mutation of sequestosome 1 (SQSTM1) in Paget's disease of bone.

Mutations affecting the ubiquitin‐associated (UBA) domain of sequestosome 1 (SQSTM1/p62) are commonly found in Pagets disease of bone (PDB) and impair SQSTM1s ability to bind ubiquitin, resulting in dysregulated NF‐κB signaling. In contrast, non‐UBA domain mutations are rarer, and little is known about how they manifest their effects. We present the first characterization at the molecular, cellular, and functional level of a non‐UBA domain missense mutation (A381V) of SQSTM1. Direct sequencing of exon 7 of the SQSTM1 gene in an Italian PDB patient detected a heterozygous C to T transversion at position 1182, resulting in an alanine to valine substitution at codon 381. Pull‐down assays showed the non‐UBA region of SQSTM1 that contains A381 is important in mediating ubiquitin‐binding affinity and that the A381V mutation exerts weak negative effects on ubiquitin binding. Structural and binding analyses of longer UBA constructs containing A381, using NMR spectroscopy and circular dichroism, showed this region of the protein to be largely unstructured and confirmed its contribution to increased ubiquitin‐binding affinity. Co‐transfections of U20S cells showed that the A381V mutant SQSTM1 co‐localized with ubiquitin with a cellular phenotype indistinguishable from wildtype. Finally, effects of the wildtype and mutant SQSTM1 on NF‐κB signaling were assessed in HEK293 cells co‐transfected with an NF‐κB luciferase reporter construct. A381V mutant SQSTM1 produced a level of activation of NF‐κB signaling greater than wildtype and similar to that of UBA domain mutants, indicating that non‐UBA and UBA domain mutations may exert their effects through a common mechanism involving dysregulated NF‐κB signaling.

Defective recognition of LC3B by mutant SQSTM1/p62 implicates impairment of autophagy as a pathogenic mechanism in ALS-FTLD

ABSTRACT Growing evidence implicates impairment of autophagy as a candidate pathogenic mechanism in the spectrum of neurodegenerative disorders which includes amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS-FTLD). SQSTM1, which encodes the autophagy receptor SQSTM1/p62, is genetically associated with ALS-FTLD, although to date autophagy-relevant functional defects in disease-associated variants have not been described. A key protein-protein interaction in autophagy is the recognition of a lipid-anchored form of LC3 (LC3-II) within the phagophore membrane by SQSTM1, mediated through its LC3-interacting region (LIR), and notably some ALS-FTLD mutations map to this region. Here we show that although representing a conservative substitution and predicted to be benign, the ALS-associated L341V mutation of SQSTM1 is defective in recognition of LC3B. We place our observations on a firm quantitative footing by showing the L341V-mutant LIR is associated with a ∼3-fold reduction in LC3B binding affinity and using protein NMR we rationalize the structural basis for the effect. This functional deficit is realized in motor neuron-like cells, with the L341V mutant EGFP-mCherry-SQSTM1 less readily incorporated into acidic autophagic vesicles than the wild type. Our data supports a model in which the L341V mutation limits the critical step of SQSTM1 recruitment to the phagophore. The oligomeric nature of SQSTM1, which presents multiple LIRs to template growth of the phagophore, potentially gives rise to avidity effects which amplify the relatively modest impact of any single mutation on LC3B binding. Over the lifetime of a neuron, impaired autophagy could expose a vulnerability, which ultimately tips the balance from cell survival toward cell death.

Insights into the Molecular Composition of Endogenous Unanchored Polyubiquitin Chains

The diverse influences of ubiquitin, mediated by its post-translational covalent modification of other proteins, have been extensively investigated. However, more recently roles for unanchored (nonsubstrate linked) polyubiquitin chains have also been proposed. Here we describe the use of ubiquitin-binding domains to affinity purify endogenous unanchored polyubiquitin chains and their subsequent characterization by mass spectrometry (MS). Using the A20 Znf domain of the ubiquitin receptor ZNF216 we isolated a protein from skeletal muscle shown by a combination of nanoLC-MS and LC-MS/MS to represent an unmodified and unanchored K48-linked ubiquitin dimer. Selective purification of unanchored polyubiquitin chains using the Znf UBP (BUZ) domain of USP5/isopeptidase-T allowed the isolation of K48 and K11-linked ubiquitin dimers, as well as revealing longer chains containing as many as 15 ubiquitin moieties, which include the K48 linkage. Top-down nanoLC-MS/MS of the A20 Znf-purified ubiquitin dimer generated diagnostic ions consistent with the presence of the K48 linkage, illustrating for the first time the potential of this approach to probe connectivity within endogenous polyubiquitin modifications. As well as providing initial proteomic insights into the molecular composition of endogenous unanchored polyubiquitin chains, this work also represents the first definition of polyubiquitin chain length in vivo.

Independent Interactions of Ubiquitin-Binding Domains in a Ubiquitin-Mediated Ternary Complex

Ubiquitin (Ub) modifications are transduced by receptor proteins that use Ub-binding domains (UBDs) to recognize distinct interaction faces on the Ub surface. We report the nuclear magnetic resonance (NMR) solution structures of the A20-like zinc finger (A20 Znf) UBD of the Ub receptor ZNF216, and its complex with Ub, and show that the binding surface on Ub centered on Asp58 leaves the canonical hydrophobic Ile44 patch free to participate in additional interactions. We have modeled ternary complexes of the different families of UBDs and show that while many are expected to bind competitively to the same Ile44 surface or show steric incompatibility, other combinations (in particular, those involving the A20 Znf domain) are consistent with a single Ub moiety simultaneously participating in multiple interactions with different UBDs. We subsequently demonstrate by NMR that the A20 Znf domain of ZNF216 and the UBA domain of the p62 protein (an Ile44-binding UBD), which function in the same biological pathways, are able to form such a Ub-mediated ternary complex through independent interactions with a single Ub. This work supports an emerging concept of Ub acting as a scaffold to mediate multiprotein complex assembly.

p62 mutations, ubiquitin recognition and Paget's disease of bone

Functional analyses of PDB (Pagets 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-kappaB (nuclear factor kappaB) 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.

Probing affinity and ubiquitin linkage selectivity of ubiquitin-binding domains using mass spectrometry.

Non-covalent interactions between ubiquitin (Ub)-modified substrates and Ub-binding domains (UBDs) are fundamental to signal transduction by Ub receptor proteins. Poly-Ub chains, linked through isopeptide bonds between internal Lys residues and the C-terminus of Ub, can be assembled with varied topologies to mediate different cellular processes. We have developed and applied a rapid and sensitive electrospray ionization-mass spectrometry (ESI-MS) method to determine isopeptide linkage-selectivity and affinity of poly-Ub·UBD interactions. We demonstrate the technique using mono-Ub and poly-Ub complexes with a number of α-helical and zinc-finger (ZnF) UBDs from proteins with roles in neurodegenerative diseases and cancer. Affinities in the 2-200 μM range were determined to be in excellent agreement with data derived from other biophysical techniques, where available. Application of the methodology provided further insights into the poly-Ub linkage specificity of the hHR23A-UBA2 domain, confirming its role in Lys48-linked poly-Ub signaling. The ZnF UBP domain of isopeptidase-T showed no linkage specificity for poly-Ub chains, and the Rabex-5 MIU also exhibited little or no specificity. The discovery that a number of domains are able to bind cyclic Lys48 di-Ub with affinities similar to those for the acyclic form indicates that cyclic poly-Ub may be capable of playing a role in Ub-signaling. Detection of a ternary complex involving Ub interacting simultaneously with two different UBDs demonstrated the co-existence of multi-site interactions, opening the way for the study of crosstalk between individual Ub-signaling pathways.

Impact of p62/SQSTM1 UBA Domain Mutations Linked to Paget’s Disease of Bone on Ubiquitin Recognition

The scaffold protein p62/SQSTM1 acts as a hub in regulating a diverse range of signaling pathways which are dependent upon a functional ubiquitin-binding C-terminal UBA domain. Mutations linked to Pagets disease of bone (PDB) commonly cluster within the UBA domain. The p62 UBA domain is unique in forming a highly stable dimer which regulates ubiquitin recognition by using overlapping surface patches in both dimerization and ubiquitin binding, making the two association events competitive. NMR structural analysis and biophysical methods show that some PDB mutations modulated the ubiquitin binding affinity by both direct and indirect mechanisms that affect UBA structural integrity, dimer stability, and contacts at the UBA-ubiquitin interface. In other cases, common PDB mutations (P392L in particular) result in no significant change in ubiquitin binding affinity for the UBA domain in isolation; however, all PDB UBA mutations lead to loss of function with respect to ubiquitin binding in the context of full-length p62, suggesting a more complex underlying mechanism.