title: A quantitative method for the specific assessment of caspase-6 activity in cell culture.
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.
authors.affiliation: Centre for Molecular Medicine and Therapeutics (CMMT), Department of Medical Genetics, CFRI, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Dagmar E
authors.initials: DE
authors.lastname: Ehrnhoefer
authors.forename: Niels H
authors.initials: NH
authors.lastname: Skotte
authors.forename: Jane
authors.initials: J
authors.lastname: Savill
authors.forename: Yen T N
authors.initials: YT
authors.lastname: Nguyen
authors.forename: Safia
authors.initials: S
authors.lastname: Ladha
authors.forename: Li-Ping
authors.initials: LP
authors.lastname: Cao
authors.forename: Edie
authors.initials: E
authors.lastname: Dullaghan
authors.forename: Michael R
authors.initials: MR
authors.lastname: Hayden
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D053178:Caspase 6 / Q000378:metabolism*; D018929:Cell Culture Techniques; D002457:Cell Extracts; D002522:Chlorocebus aethiops; D057075:Enzyme Assays / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D007700:Kinetics; D034882:Lamins / Q000378:metabolism; D049449:Luminescence; D051379:Mice; D008969:Molecular Sequence Data; D009474:Neurons / Q000201:enzymology; D010455:Peptides / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: D002457:Cell Extracts; D034882:Lamins; D010455:Peptides; D053178:Caspase 6