Detection of E2 Activity as a Tumor Marker using an Artificial Ring Finger

Abstract

Protein ubiquitination is undertaken through an enzymatic cascade comprising ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. The E2 enzymes are associated with various cancers such as leukemia, lung cancer, and breast cancer, and thus, they are attractive targets as tumor markers. In a previous study, we reported the creation of artificial RING fingers that are useful for detecting E2 activity. It was recently reported that a signal accumulation ISFET biosensor (AMIS sensor) allows the detection of proton changes arising during enzyme reactions. In this study, by using the artificial RING fingers and the AMIS sensor, we have succeeded in high-sensitivity quantitative detection of E2 activity. Furthermore, the real-time capturing of E2 activity was successfully achieved using human acute promyelocytic leukemia cells after treatment with the anticancer drug bortezomib. This study allowed the assessment of the pathological conditions of cancer cells. The present method could be applicable to the detection of E2 activity in the blood serum of patients, and therefore, it could be helpful for designing suitable treatment programs for patients. This strategy is extremely simple and convenient, and it opens new possibilities for diagnostic and prognostic methods in tumor development. Patients with cancer could benefit from advances of the quantitative analysis of the E2 activities of the ubiquitination system.