Diablo ubiquitination analysis by sandwich immunoassay

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Biology

Background:

• Ubiquitin system deregulation is implicated in various human diseases.
• Inhibitors of Apoptosis Proteins (IAPs) regulate apoptosis by targeting caspases.
• Diablo acts as a negative regulator of IAPs in the apoptotic pathway.

Purpose of the Study:

• To develop a sensitive assay for quantifying Diablo ubiquitination.
• To identify inhibitors of the IAP ubiquitination cascade for drug discovery.
• To evaluate the performance of the developed assay panel.

Main Methods:

• Development of sandwich immunoassays utilizing an electrochemical luminescence (ECL) platform.
• Quantification of total Diablo, ubiquitinated Diablo, and K48-specific ubiquitinated Diablo.
• Detection of Diablo ubiquitination level changes in response to IAP or proteasome inhibitors.

Main Results:

• The ECL-based assay panel successfully quantifies Diablo ubiquitination.
• The assay detects changes in ubiquitination in the presence of inhibitors.
• The assay panel demonstrated good precision, linearity, and limit of quantification.

Conclusions:

• The developed ECL assay is a cost-efficient, high-throughput method for drug discovery.
• This assay facilitates the study of IAP ubiquitination cascade.
• The assay has potential for identifying novel IAP inhibitors.