A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture Dagmar E. Ehrnhoefer1, Niels H. Skotte1,3, Jane Savill2, Yen T. N. Nguyen1, Safia Ladha1, Li-Ping Cao1, Edie Dullaghan2, Michael R. Hayden1* 1 Centre for Molecular Medicine and Therapeutics (CMMT), Department of Medical Genetics, CFRI, University of British Columbia, Vancouver, British Columbia, Canada, 2 Centre for Drug Research and Development (CDRD), Vancouver, British Columbia, Canada, 3 Institute of Cellular and Molecular Medicine (ICMM), Department of Medical Genetics, University of Copenhagen, Copenhagen, Denmark 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. Citation: Ehrnhoefer DE, Skotte NH, Savill J, Nguyen YTN, Ladha S, et al. (2011) A Quantitative Method for the Specific Assessment of Caspase-6 Activity in Cell Culture. PLoS ONE 6(11): e27680. doi:10.1371/journal.pone.0027680 Editor: Andrea C. LeBlanc, McGill University, Canada Received June 10, 2011; Accepted October 21, 2011; Published November 29, 2011 Copyright: 2011 Ehrnhoefer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Canadian Institutes of Health Research (CIHR MOP-84438) and the Cure Huntington Disease Initiative (CHDI) ‘TREAT HD’ grants to MRH. DEE is a CIHR fellowship awardee and held a postdoctoral fellowship of the Austrian Science Fund (FWF), project no. J2797-B09. MRH is a University Killam Professor and holds a Canada Research Chair in Human Genetics. The authors gratefully acknowledge partial funding by the Pfizer-CDRD Innovation Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The Pfizer Innovation fund funded part of the work that was done at the Centre for Drug Research and Development (CDRD). The authors do not have any employment or consultant relationships with Pfizer, there are no relevant patents or products. The authors confirm that the funding does not alter their adherence to the PLos ONE policies on sharing data and materials. * E-mail: mrh@cmmt.ubc.ca 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). This definition however has become somewhat inaccurate as an increasing number of non-apoptotic roles for both initiator and executioner caspases have been identified that mediate cell differentiation, maturation and signaling events [1]. Caspases can further be distinguished based on their inherent differences in caspase substrate preference that are defined by the shape and electrostatic potential of the active site cleft [2]. 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]. However, with Km/kcat ratio differences of less than 10 fold for many widely used peptide substrates [4], these reagents are not particularly useful for investigating the activity of a caspase present at lower concentrations in cell culture and tissue samples. 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. In biologic protease substrates, additional factors outside the 4 amino acid recognition site can influence the selectivity and efficiency of proteolytic cleavage. For caspases, it has been shown that the amino acid residue directly after the scissile bond (P19) is an important determinant of cleavage, since charged or bulky residues are not well tolerated [7]. Furthermore, domains far away from the cleavage site can mediate the interaction between substrate and protease (exosites), and although such interactions have not yet been shown for proteases of the caspase family, the high variability of cleavage site motifs in natural caspase substrates argues in favour of the presence of exosites. Known substrates for caspase-6 show a particularly high variability in their recognition sequences [8], with cleavage sites other than I/D/E/L/T/V, E/ D/Q, X, D found in substrates such as the presenilins (ENDD, PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27680