Characterizing polyubiquitinated forms of the neurodegenerative ubiquitin mutant UBB⁺¹

The ubiquitin mutant UBB⁺¹ has been identified as a hallmark of neurodegenerative diseases. In this study, we characterize polyubiquitinated forms of UBB⁺¹ in vitro and from patient samples. The ability of UBB⁺¹ to be readily ubiquitinated by several E2 enzymes provided a mechanism for the controlled synthesis and purification of defined conjugates. This allowed us to utilize polyUb-UBB⁺¹ conjugates for biochemical assays, as well as solution NMR. Coupled with our immunoassay for detection of ubiquitinated forms of UBB⁺¹ in patient blood samples, we gain a clearer picture of the molecular mechanisms underlying neurodegenerative diseases.