Development of Ubiquitin Variants with Selectivity for Ubiquitin C-Terminal Hydrolase Deubiquitinases
| Autor: | Chittaranjan Das, Chad S. Hewitt, Daniel P. Flaherty, Aaron D Krabill |
|---|---|
| Rok vydání: | 2020 |
| Předmět: |
Mutation
biology Ubiquitin Chemistry Ubiquitination Ubiquitin C-Terminal Hydrolase medicine.disease_cause Biochemistry Protein ubiquitination Deubiquitinating enzyme Cell biology Hydrolase biology.protein medicine Transcriptional regulation Humans Protein Processing Post-Translational Ubiquitin Thiolesterase Deubiquitination |
| Zdroj: | Biochemistry. 59:3447-3462 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/acs.biochem.9b01076 |
| Popis: | Ubiquitin (Ub) is a highly conserved protein that is covalently attached to substrate proteins as a post-translational modification to regulate signaling pathways such as proteasomal degradation and cell cycle/transcriptional regulation in the eukaryotic cellular environment. Ub signaling is regulated by the homeostasis of substrate protein ubiquitination/deubiquitination by E3 ligases and deubiquitinating enzymes (DUBs) in healthy eukaryotic systems. One such DUB, ubiquitin C-terminal hydrolase L1 (UCHL1), is endogenously expressed in the central nervous system under normal physiological conditions, but overexpression and/or mutation has been linked to various cancers and neurodegenerative diseases. The lack of UCHL1 probing strategies suggests development of a selective Ub variant (UbV) for probing UCHL1's role in these disease states would be beneficial. We describe a computational design approach to investigate UbVs that lend selectivity, both binding and inhibition, to UCHL1 over the close structural homologue UCHL3 and members of other DUB families. A number of UbVs, mainly those containing Thr9 mutations, displayed appreciable binding and inhibition selectivity for UCHL1 over UCHL3, compared to wild-type Ub in in vitro assays. By appending reactive electrophiles to the C-terminus of the UbVs, we created the first activity-based probe (ABP) with demonstrated reaction selectivity for UCH family DUBs over other families in cell lysates. Further kinetic analysis of covalent inhibition by the UbV-ABP with UCHL1 and UCHL3 offers insight into the future design of UCHL1 selective UbV-ABP. These studies serve as a proof of concept of the viability of the in silico design of ubiquitin variants for UCH family DUBs as a step toward the development of macromolecular UCHL1 inhibitors. |
| Databáze: | OpenAIRE |
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