A Whole Cell Assay to Measure Caspase-6 Activity by Detecting Cleavage of Lamin A/C

Caspase-6 is a cysteinyl protease implicated in neurodegenerative conditions including Alzheimer's and Huntington's disease making it an attractive target for therapeutic intervention. A greater understanding of the role of caspase-6 in disease has been hampered by a lack of suitable cellular assays capable of specifically detecting caspase-6 activity in an intact cell environment. This is mainly due to the use of commercially available peptide substrates and inhibitors which lack the required specificity to facilitate development of this type of assay. We report here a 384-well whole-cell chemiluminescent ELISA assay that monitors the proteolytic degradation of endogenously expressed lamin A/C during the early stages of caspase-dependent apoptosis. The specificity of lamin A/C proteolysis by caspase-6 was demonstrated against recombinant caspase family members and further confirmed in genetic deletion studies. In the assay, plasma membrane integrity remained intact as assessed by release of lactate dehydrogenase from the intracellular environment and the exclusion of cell impermeable peptide inhibitors, despite the induction of an apoptotic state. The method described here is a robust tool to support drug discovery efforts targeting caspase-6 and is the first reported to specifically monitor endogenous caspase-6 activity in a cellular context

Effect of peptide-based caspase inhibitors on SKNAS cells as determined using the Caspase-Glo® 6 assay.

<p>SKNAS cells were treated with Ac-VEID-CHO (•) or Ac-DEVD-CHO (▪) prior to addition of 3 µM staurosporine for 6 hours and detection of VEID-ase activity as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.</p

Comparison of potency of peptide-derived caspase inhibitors in cellular lamin cleavage assay, enzymatic activity and cell permeability.

<p>nd = not determined</p><p>a = z-VEID-TFPM enzymatic IC₅₀ is less than 0.0017 due to limit of enzymatic assay detection.</p><p>*Enzymatic IC₅₀ values were determined after a 15 minute enzyme/inhibitor preincubation and 40 minute enzyme reaction.</p

Effect of peptide-based caspase inhibitors on the generation of cleaved lamin A/C in SKNAS cells.

<p>(A) SKNAS cells were treated with z-VEID-FMK (♦), z-DEVD-FMK (▪), Q-VD-OPh (•), Ac-VEID-CHO (▴) or Ac-DEVD-CHO (○) prior to addition of 3 µM staurosporine for 6 hours. (B) SKNAS cells were treated with z-ID-TFPM (•), z-EID-TFPM (▴) or z-VEID-TFPM (▪) prior to addition of 3 µM staurosporine for 6 hours. Detection of the small lamin A/C cleavage product was performed as described in Experimental Procedures. Concentration inhibition curves were performed in duplicate and represent 1 of at least 3 experiments with similar results. Concentration-response curves for each inhibitor were normalized to zero and 100% based on no staurosporine or DMSO, respectively. The mean and standard error of the mean are reported.</p

Apoptosis-mediated cleavage of lamin A/C is elevated in wild-type relative to caspase-6 KO fibroblasts.

<p>Fibroblasts derived from caspase-6 KO (▪) or wild type (•) mice were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product. The assay was performed in quadruplicate two times with similar results; mean and standard error of the mean are reported.</p

Potency of peptide-derived caspase inhibitors possessing aldehyde (CHO) and fluoromethyl ketone (FMK) warheads against executioner caspases.

<p>*IC₅₀ values were determined after a 15 minute enzyme/inhibitor preincubation and 40 minute enzyme reaction. Due to the capacity for time-dependent inhibition with irreversible inhibitors the values reported can be considered “apparent IC₅₀”.</p

Effect of staurosporine on the generation of cleaved lamin A/C in SKNAS cells.

<p>SKNAS cells were treated with the indicated concentration of staurosporine for 6 hours prior to detection of the small lamin A/C cleavage product as described in Experimental Procedures. The assay was performed in triplicate one time. The mean and standard error of the mean are reported.</p

Western blot detection of lamin A/C from SKNAS neuroblastoma cells upon staurosporine treatment.

<p>(A) Schematic of the N- and C-terminal globular domains of Lamin A/C with VEID-containing central α-helical region as the site of caspase-6 proteolysis. (B) SKNAS cells were treated with DMSO control or staurosporine for 6 hours prior to cell lysis. Lysates were probed for small lamin A/C subunit (Lanes 1–2), large lamin A/C subunit (Lanes 3–4) or total lamin A/C (Lanes 5–6). (C) SKNAS cells were treated with DMSO or staurosporine for the indicated time prior to cell lysis. Lysates were probed for large lamin A/C subunit (red) or β-Actin (green).</p

Western blot detection of recombinant GST-lamin A processing by purified caspases.

<p>(A) The indicated concentration of caspase-6 was incubated with GST-lamin A for two hours. (B) 300 Units of caspases 1–9 were incubated with GST-lamin A for two hours. Intact and cleaved lamin A were detected via western blotting using anti-GST antibody.</p

Effect of staurosporine on the release of Lactate Dehydrogenase in SKNAS cells.

<p>SKNAS cells were treated with the indicated concentration of staurosporine for 2 (•), 4 (▪), 6 (▴) or 8 (♦) hours prior to detection of LDH release to the cell supernatant. The assay was performed in triplicate and represents 1 of at least 2 experiments with similar results. The data was normalized to fold increase over DMSO treatment. The mean and standard error of the mean are reported.</p