Method for measuring the activity of deubiquitinating enzymes in cell lines and tissue samples.

The ubiquitin-proteasome system has recently been implicated in various pathologies including neurodegenerative diseases and cancer. In light of this, techniques for studying the regulatory mechanism of this system are essential to elucidating the cellular and molecular processes of the aforementioned diseases. The use of hemagglutinin derived ubiquitin probes outlined in this paper serves as a valuable tool for the study of this system. This paper details a method that enables the user to perform assays that give a direct visualization of deubiquitinating enzyme activity. Deubiquitinating enzymes control proteasomal degradation and share functional homology at their active sites, which allows the user to investigate the activity of multiple enzymes in one assay. Lysates are obtained through gentle mechanical cell disruption and incubated with active site directed probes. Functional enzymes are tagged with the probes while inactive enzymes remain unbound. By running this assay, the user obtains information on both the activity and potential expression of multiple deubiquitinating enzymes in a fast and easy manner. The current method is significantly more efficient than using individual antibodies for the predicted one hundred deubiquitinating enzymes in the human cell.

Small-molecule RA-9 inhibits proteasome-associated DUBs and ovarian cancer in vitro and in vivo via exacerbating unfolded protein responses.

PURPOSE: Ovarian cancer is the deadliest of the gynecologic malignancies. Carcinogenic progression is accompanied by upregulation of ubiquitin-dependent protein degradation machinery as a mechanism to compensate with elevated endogenous proteotoxic stress. Recent studies support the notion that deubiquitinating enzymes (DUB) are essential factors in proteolytic degradation and that their aberrant activity is linked to cancer progression and chemoresistance. Thus, DUBs are an attractive therapeutic target for ovarian cancer. EXPERIMENTAL DESIGN: The potency and selectivity of RA-9 inhibitor for proteasome-associated DUBs was determined in ovarian cancer cell lines and primary cells. The anticancer activity of RA-9 and its mechanism of action were evaluated in multiple cancer cell lines in vitro and in vivo in immunodeficient mice bearing an intraperitoneal ES-2 xenograft model of human ovarian cancer. RESULTS: Here, we report the characterization of RA-9 as a small-molecule inhibitor of proteasome-associated DUBs. Treatment with RA-9 selectively induces onset of apoptosis in ovarian cancer cell lines and primary cultures derived from donors. Loss of cell viability following RA-9 exposure is associated with an unfolded protein response as mechanism to compensate for unsustainable levels of proteotoxic stress. In vivo treatment with RA-9 retards tumor growth, increases overall survival, and was well tolerated by the host. CONCLUSIONS: Our preclinical studies support further evaluation of RA-9 as an ovarian cancer therapeutic.