Design of a System for Monitoring Ubiquitination Activities of E2 Enzymes Using Engineered RING Finger Proteins

Methods in Molecular Biology
Kazuhide Miyamoto, Kazuki Saito

Abstract

Ubiquitination is a sequential cascade consisting of ubiquitin-activating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligating (E3) enzymes. It controls numerous processes such as protein degradation, DNA repair, and signal transduction pathways. E2 enzymes are associated with a variety of diseases such as leukemia, breast cancer, lung cancer, and colorectal cancer. To date, the monitoring of E2 activity for cancer diagnosis is challenging due to its intricate cascade reaction. To surmount this hurdle, we have recently developed a novel strategy for monitoring E2 activities. Here, we describe the concise machinery of ubiquitination with artificial RING finger proteins (ARFs) functioning as E3 enzymes. This machinery enables the simplified monitoring of E2 activities. Furthermore, our system combines a signal accumulation ion-sensitive field-effect transistor biosensor with ARFs, allowing for real-time monitoring of the pathological conditions of cancer cells. The present methodology may lead to novel diagnostic techniques for cancers.

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Biosensors for Cancer Detection

Biosensors are devices that are designed to detect a specific biological analyte by essentially converting a biological entity (ie, protein, DNA, RNA) into an electrical signal that can be detected and analyzed. The use of biosensors in cancer detection and monitoring holds vast potential. Biosensors can be designed to detect emerging cancer biomarkers and to determine drug effectiveness at various target sites. Biosensor technology has the potential to provide fast and accurate detection, reliable imaging of cancer cells, and monitoring of angiogenesis and cancer metastasis, and the ability to determine the effectiveness of anticancer chemotherapy agents.

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