Non-caspase proteases play an important role in cell death.
Inhibition of caspases after mitochondrial dysfunction only results in a delay and not in complete inhibition of apoptosis, highlighting the essential role of other cysteine proteases such as calpains and cathepsins. These non-caspase proteases have been long associated with necrotic cell death, and it is only recently that they have been associated with apoptosis.
Calpains are cytosolic calcium-dependent cysteine proteases composed of one or two subunits. Calpain cleavage sites are not sequence-specific, and tertiary structure elements rather than primary amino acid sequences seem to be responsible for directing cleavage to a specific site.
A broad range of proteins can be cleaved by calpain, including cytoskeletal proteins such as α-fodrin, ion channels, growth factor receptors and adhesion molecules. Calpain activation has been implicated in neuronal apoptosis in ischemic brain injury and neurodegenerative diseases such as Alzheimer’s.
Calpain activity can be easily detected in many cell types using a specific calpain substrate linked to a colorimetric or fluorogenic detection molecule that will be released upon cleavage of the substrate.
There are few considerations to keep in mind when investigating apoptosis-related calpain activity:
Cathepsins are proteases found in all animals. While most cathepsins are cysteine proteases (cathepsins B, F, K, L and S), cathepsin D is an aspartic protease and cathepsin G a serine protease. Cathepsins are generally found in lysosomes. They become activated at the low pH found in this organelle, and thus have been historically associated with necrosis. Some cathepsins remain active at neutral pH (in the cytosol), and are associated with apoptosis signals such as caspase 8 activation through TNF alpha.
Like caspase and calpain activity, cathepsin activity can be easily detected in many cell types using a specific cathepsin substrate linked to a colorimetric or fluorogenic detection molecule, which will be released upon cleavage of the substrate.
There are few considerations to keep in mind when investigating apoptosis-related cathepsin activity:
Cathepsin B activity assay kit (fluorometric) ab65300: Quantification of basal Cathepsin B activity in HL60 cell lysates in absence or presence of Cathepsin B inhibitor.