Functional interaction between sequestosome-1/p62 and autophagy-linked FYVE-containing protein WDFY3 in human osteoclasts

Abstract

Paget's disease of bone (PDB) is a late-onset disorder characterised by focal areas of increased bone resorption, with osteoclasts that are increased in size, multinuclearity, number and activity. PDB-causing missense and nonsense variants in the gene encoding Sequestosome-1/p62 (SQSTM1) have been identified, all of which cluster in and around the ubiquitin-associated (UBA) domain of the protein. SQSTM1 is ubiquitously expressed and there is, as yet, no clear reason why these mutations only appear to cause an osteoclast-related phenotype. Using co-immunoprecipitation and tandem mass spectrometry, we identified a novel interaction in human osteoclast-like cells between SQSTM1 and Autophagy-Linked FYVE domain-containing protein (ALFY/WDFY3). Endogenous ALFY and SQSTM1 both localised within the nuclei of osteoclasts and their mononuclear precursors. When osteoclasts were starved to induce autophagy, SQSTM1 and ALFY relocated to the cytoplasm where they formed large aggregates, with cytoplasmic relocalisation appearing more rapid in mature osteoclasts than in precursors in the same culture. Overexpression of wild-type SQSTM1 in HEK293 cells also resulted in the formation of cytoplasmic aggregates containing SQSTM1 and endogenous ALFY, as did overexpression of a PDB-causing missense mutant form of SQSTM1, indicating that this mutation does not impair the formation of SQSTM1- and ALFY-containing aggregates. Expression of ALFY in bone cells has not previously been reported, and the process of autophagy has not been studied with respect to osteoclast activity. We have identified a functional interaction between SQSTM1 and ALFY in osteoclasts under conditions of cell stress. The difference in response to starvation between mature osteoclasts and their precursors may begin to explain the cell-specific functional effects of SQSTM1 mutations in PDB.