Seminars

Understanding the turnover of the truncated form of mutant ubiquitin UBB⁺¹, UBB^G76Y

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 (UB^G76Y). Lacking a C-terminal glycine at position 76, UB^G76Y is unable to conjugate to substrates. To date, little is known about UB^G76Y. We wanted to study the fate of UB^G76Y 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 UB^G76Y in HEK293 cells and observed that UB^G76Y does not accumulate  to the same extent as UBB⁺¹. In addition, by inhibiting degradation pathways we provide evidence that UB^G76Y is degraded by autophagy. To summarize, converting UBB⁺¹ to UB^G76Y may be a strategy of cells to alleviate the stress associated with UBB⁺¹ .