James R. Cavey

Novel UBA Domain Mutations of SQSTM1 in Paget's Disease of Bone: Genotype Phenotype Correlation, Functional Analysis, and Structural Consequences

Three novel missense mutations of SQSTM1 were identified in familial PDB, all affecting the UBA domain. Functional and structural analysis showed that disease severity was related to the type of mutation but was unrelated to the polyubiquitin‐binding properties of the mutant UBA domain peptides.

Structure of the Ubiquitin-associated Domain of p62 (SQSTM1) and Implications for Mutations That Cause Paget's Disease of Bone

The p62 protein (also known as SQSTM1) mediates diverse cellular functions including control of NFκB 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 Pagets 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.

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 associated with Paget's disease of bone p62 (SQSTM1) mutations.

We have studied the effects of various PDB‐causing mutations of SQSTM1 on the in vitro ubiquitin‐binding properties of the p62 protein. All mutations caused loss of monoubiquitin‐binding and impaired K48‐linked polyubiquitin‐binding, which was only evident at physiological temperature. This suggests that SQSTM1 mutations predispose to PDB through a common mechanism that depends on loss of ubiquitin‐binding by p62.

Role of ubiquitin-mediated proteolysis in the pathogenesis of neurodegenerative disorders

Intraneuronal inclusions containing ubiquitylated filamentous protein aggregates are a common feature of many of the major human neurodegenerative disorders, including Alzheimers and Parkinsons disease. Loss of function mutations in enzymes of the ubiquitin conjugation/deconjugation pathway are sufficient to cause familial forms of neurodegenerative diseases, suggesting that failure of ubiquitin-mediated proteolysis could also be central to inclusion formation in the more common sporadic cases. Examination of ubiquitin-positive inclusions at the protein level provides evidence of attempted proteasomal proteolysis, however close inspection of the temporal aspects of inclusion formation indicates that ubiquitylation is probably a late event. In this regard, the presence of ubiquitin within inclusions of idiopathic neurodegenerative disorders may indicate not a primary dysfunction of ubiquitin-mediated proteolysis, but rather a secondary, presumably protective cellular response. Within this model, other factors are likely to be initiating in inclusion biogenesis. Consistent with these proposals, non-ubiquitylated forms of the principal ubiquitylated components of Alzheimers disease neurofibrillary tangles and Parkinsons disease Lewy bodies, tau and alpha-synuclein proteins, respectively, can be degraded by proteasomes in a pathway which does not have an absolute requirement for ubiquitylation. Inhibition of proteasome function in the pathological state, as has been reported in both Alzheimers and Parkinsons disease, could therefore contribute both to accumulation of non-ubiquitylated forms of aggregation-prone neuronal proteins, as well as impaired clearance of ubiquitylated aggregates.

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.

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.

Structural and functional studies of mutations affecting the UBA domain of SQSTM1 (p62) which cause Paget's disease of bone

Mutations affecting the UBA (ubiquitin-associated) domain of SQSTM1 (Sequestosome 1) (p62) are a common cause of Pagets 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.

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.

Immunoreactivity to Lys63-linked polyubiquitin is a feature of neurodegeneration

The major human neurodegenerative diseases are characterised by ubiquitin-positive intraneuronal inclusions, however the precise nature of the ubiquitin modifications in these structures is unclear. Using a monoclonal antibody specific for Lys63-linked polyubiquitin we have performed the first immunohistochemical analysis of linkage-specific ubiquitination in vivo associated with neurodegeneration. Immunoreactivity was detected within the pathological lesions of Alzheimers, Huntingtons and Parkinsons disease brains, although staining of Lewy bodies in the substantia nigra in Parkinsons disease was rare, indicating a selective involvement of Lys63-linked polyubiquitin in inclusion biogenesis in this disorder. Immunoreactivity was also a feature in neurons of proteasome-depleted mice, suggesting a proteasomal contribution to the degradation of Lys63-linked polyubiquitinated proteins in vivo.