A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture

Dagmar E Ehrnhoefer,Yen T N Nguyen,Edie Dullaghan,Niels H Skotte,Safia Ladha,Michael R Hayden,Li-Ping Cao,Jane Savill,Andrea C Leblanc

doi:10.1371/journal.pone.0027680

Abstract

Aberrant activation of caspase-6 has recently emerged as a major contributor to the pathogeneses of neurodegenerative disorders such as Alzheimer's and Huntington disease. Commercially available assays to measure caspase-6 activity commonly use the VEID peptide as a substrate. However these methods are not well suited to specifically assess caspase-6 activity in the presence of other, confounding protease activities, as often encountered in cell and tissue samples. Here we report the development of a method that overcomes this limitation by using a protein substrate, lamin A, which is highly specific for caspase-6 cleavage at amino acid 230. Using a neo-epitope antibody against cleaved lamin A, we developed an electrochemiluminescence-based ELISA assay that is suitable to specifically detect and quantify caspase-6 activity in highly apoptotic cell extracts. The method is more sensitive than VEID-based assays and can be adapted to a high-content imaging platform for high-throughput screening. This method should be useful to screen for and characterize caspase-6 inhibitor compounds and other interventions to decrease intracellular caspase-6 activity for applications in neurodegenerative disorders.

Highlights

Proteases of the caspase family are known as important mediators of apoptosis and have been commonly subdivided based on their roles in apoptosis or inflammation
In order to develop an activity assay that is more specific for caspase-6 than available methods relying on the cleavage of the VEID peptide, we decided to investigate the cleavage of known caspase-6 substrates
The study of the role of caspases during apoptosis, and during non-apoptotic developmental and signalling processes, is hampered by the lack of specific assays to measure the activity of single members of the caspase family

Summary

Introduction

Proteases of the caspase family are known as important mediators of apoptosis and have been commonly subdivided based on their roles in apoptosis or inflammation (apoptotic initiator, apoptotic executioner or inflammatory caspases). Using positional scanning of peptide libraries, consensus recognition sequences have been proposed for each caspase and have led to the development of peptide substrates as well as inhibitors that typically consist of 4 amino acids (i.e. DEVD for caspase-3), followed by a fluorescent tag such as Afc (7-amino-4-trifluoro methylcoumarin) for a substrate or a ‘warhead’ such as fmk (fluoromethylketone) that covalently binds the enzyme for an inhibitor. These reagents are useful to investigate caspases that constitute the majority of caspase-like activity in a sample, as it may be assumed for active caspase-3 in highly apoptotic extracts [3]. In particular in developmental or signalling processes that do not involve cell death, intracellular caspase activity is likely under tight control by endogenous caspase inhibitors or the proteasome [5,6] and the resulting low levels of activity are difficult to detect with peptide substrates

Methods

Results

Conclusion