Polyubiquitin linkage profiles in three models of proteolytic stress suggest the etiology of alzheimer disease

Polyubiquitin chains on substrates are assembled through any of seven lysine residues or the N terminus of ubiquitin (Ub), generating diverse linkages in the chain structure. PolyUb linkages regulate the fate of modified substrates, but their abundance and function in mammalian cells are not well studied.We present a mass spectrometry-based method to measure polyUb linkages directly from total lysate of mammalian cells. In HEK293 cells, the level of polyUb linkages was found to be 52% (Lys⁴⁸), 38% (Lys⁶³), 8% (Lys²⁹), 2% (Lys¹¹), and 0.5% or less for linear, Lys⁶, Lys²⁷, and Lys³³ linkages. Tissue specificity of these linkages was examined in mice fully labeled by heavy stable isotopes (i.e. SILAC mice). Moreover, we profiled the Ub linkages in brain tissues from patients of Alzheimer disease with or without concurrent Lewy body disease as well as three cellular models of proteolytic stress: proteasome deficiency, lysosome deficiency, and heat shock. The data support that polyUb chains linked through Lys⁶, Lys¹¹, Lys²⁷, Lys²⁹, and Lys⁴⁸ mediate proteasomal degradation, whereas Lys⁶³ chains are preferentially involved in the lysosomal pathway. Mixed linkages, including Lys⁴⁸, may also contribute to lysosomal targeting, as both Lys⁶³ and Lys⁴⁸ linkages are colocalized in LC3-labeled autophagosomes. Interestingly, heat shock treatment augments Lys¹¹, Lys⁴⁸, and Lys⁶³ but not Lys²⁹ linkages, and this unique pattern is similar to that in the profiled neurodegenerative cases. We conclude that different polyUb linkages play distinct roles under the three proteolytic stress conditions, and protein folding capacity in the heat shock responsive pathway might be more affected in Alzheimer disease.