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.
| Original language | English |
|---|---|
| Journal | PLOS ONE |
| Volume | 6 |
| Issue number | 11 |
| Pages (from-to) | e27680 |
| ISSN | 1932-6203 |
| DOIs | |
| Publication status | Published - 29 Nov 2011 |
Keywords
- Amino Acid Sequence
- Animals
- COS Cells
- Caspase 6
- Cell Culture Techniques
- Cell Extracts
- Cercopithecus aethiops
- Enzyme Assays
- Enzyme-Linked Immunosorbent Assay
- Fluorescent Antibody Technique
- Humans
- Kinetics
- Lamins
- Luminescence
- Mice
- Molecular Sequence Data
- Neurons
- Peptides
- Substrate Specificity