Development of a general method to assay deubiquitinase activities

Teagen Hartley, Jordan Brooks, Aidan Baker, Olivia Clarke, Vincent Leahy, Eden Erazua, and Yun-Seok Choi,,,,, School of Natural Sciences, 1200 University St, Unit 9008, Black Hills State University, Spearfish, South Dakota, 57799-9008

Deubiquitinatase (DUB) is an enzyme responsible for the removal of ubiquitin (Ub) from proteins. The human genome encodes nearly 100 DUBs playing important roles in cell cycle control, DNA damage repair, and disease pathways. Deregulation of DUBs impact on human disease, particularly cancer, neurodegeneration, and inflammation, which highlights the therapeutic potential for pharmacological modulation of DUB activities. Although DUB activities are essential information to understand DUB mechanism and to develop drugs, there is no general method to assay DUB activities with physiological substrates. Sensors have recently been developed to allow monitoring DUB activities. DUB activities are generally determined by titrating low (10 nanomolar) to high (100 micromolar, µM) concentration of targets. However, the sensors start to bind off-targets when a target concentration is more than 10 µM, which makes the DUB assays impractical. We need a new sensor to assay DUB activities because most DUB assays require a titration with more than 10 µM of target concentration.

We modeled the sensor structure binding to the off-target. Based upon the modeling, we chose one of the key amino acids in the sensor responsible for binding to the off target and mutated it to weaken the sensor’s binding affinity to the off-target. The designed protein sensor with the mutation, tUI2, was prepared. tUI2 was labeled with Atto532 dye for the quantification of free Ub. The binding affinity of Atto532-tUI2 to free Ub, an on-target of the sensor, is 1.2 nM Kd which means that the sensor still detects its on-target sensitively. We will further test the Atto532-tUI2 binding affinity for its off-target to determine whether Atto532-tUI2 can be used as a sensor for DUB assays.

Additional Abstract Information

Presenters: Teagen Hartley, Teagen Hartley, Jordan Brooks, Olivia Clarke, Aidan Baker, Eden Erazua, Vincent Leahy

Institution: Black Hills State University

Type: Poster

Subject: Chemistry

Status: Approved

Time and Location

Session: Poster 4
Date/Time: Tue 11:00am-12:00pm
Session Number: 3584