Understanding the turnover of the truncated form of mutant ubiquitin UBB+1, UBBG76Y
Ubiquitin is a 76 amino acid molecule that labels proteins for degradation. Its C-terminus at 76 glycine is conjugated to the side chain of a substrate lysine. The only known ubiquitin mutant is ubiquitin B+1 (UBB+1), caused by a non-heredity missense mutation during transcription of the UBB gene. The resulting protein is a ubiquitin domain with a G76Y mutation and an additional 19 amino acids (AA) tail. This UBB+1 protein is found in sporadic and familial AD brains. It was recently shown in our lab that the expression of UBB+1 accelerates AD pathology. UBB+1 has been claimed to be cleaved by a carboxyl-terminal hydrolase isozyme L3 (UCHL3), resulting in a truncated form of UBB+1: ubiquitin with a G76Y mutation (UBG76Y). Lacking a C-terminal glycine at position 76, UBG76Y is unable to conjugate to substrates. To date, little is known about UBG76Y. We wanted to study the fate of UBG76Y and understand whether it alleviates or exacerbates the stress imposed by UBB+1. We generated a UCHL3 KO cell line in HEK293 using CRISPR/Case9 and showed that UCHL3 is the main enzyme that cleaves UBB+1 in mammalian cells. We also expressed UBG76Y in HEK293 cells and observed that UBG76Y does not accumulate to the same extent as UBB+1. In addition, by inhibiting degradation pathways we provide evidence that UBG76Y is degraded by autophagy. To summarize, converting UBB+1 to UBG76Y may be a strategy of cells to alleviate the stress associated with UBB+1 .