Non-caspase proteases are important players in bringing about programmed cell death. If caspases are inhibited following mitochondrial dysfunction, apoptosis is not abolished, but rather delayed. This shows that other cysteine proteases are vital to this process, such as cathepsins and calpains. They have been known to accompany necrosis of cells for a long time, but only recently has it come to light that their presence is associated with apoptosis.
Calpains are cysteine proteases located in the cytosol and are dependent upon calcium for their activity. They have either one or two subunits. Their cleavage sites do not rely on specific sequences of the primary amino acids, and instead cleavage occurs at specific sites based on certain elements of the tertiary structure.
Calpains can cleave a wide array of proteins such as those found in the cytoskeleton, like alpha-fodrin, ion channels, receptors for growth factor and cell adhesion molecules. The activation of calpains is found to be associated with the apoptosis of nerve cells following ischemic injury to the brain as well as in diseases caused by neurodegeneration, such as Alzheimer’s.
It is possible to detect the presence of calpain quite easily in a variety of cells by a specific calpain substrate that is conjugated to a detector or indicator molecule of either colorimetric or fluorogenic capability. This is activated upon substrate cleavage.
Some factors must be remembered when calpain activity is analyzed in relation to apoptosis:
Avoid all contamination by protease-rich lysosomes or any other cell component which is capable of providing a false positive. It may even be required that lysosomal protease (cathepsin) inhibitors be used to restrict the scope of false positives due to non-specific protease activity.
Another recommendation is the use of generic caspase inhibitors to distinguish the various cysteine proteases.
Cells must be treated with calpain-specific inhibitors to provide negative controls so that the activity that is measured is verified to be due to calpain cleavage.
There is always some level of calpain activity within a cell, which must be measured at baseline by using appropriate untreated cells as the background controls.
The mere demonstration of calpain activity does not indicate that apoptosis is occurring. Calpain also has other modes of activity including activation during cell necrosis. This is why appropriate controls must be used, and why other parameters of apoptosis that apply to the relevant situation are considered.
Calpain activity assay (ab65308): Calpain activity measured in Jurkat cells in the absence (naïve) or presence of 10 μM Camptothecin (CPT) or 10 μg/mL Cycloheximide (CHX) for 4 hours.
Cathepsins are proteases found almost universally. They are typically cysteine proteases, including cathepsins B, F, K, L and S. However, cathepsin D and cathepsin G are aspartic and serine proteases respectively. These enzymes are lysosomal, and become active at the low pH associated with these cell organelles. For this reason they have been linked to apoptosis from a historical perspective. Some do show activity at a neutral pH, usually within the cytosol. These are usually found in association with signals of apoptosis such as caspase 8 activation via TNF-α.
Similar to the activity of both caspases and calpain, it is easy to detect the activity of cathepsin over a wide range of cell types. This uses a specific substrate for the cathepsin which is conjugated to a detector molecule that shows colorimetric or fluorogenic change on substrate cleavage.
Some things to consider when testing for cathepsin activity in relation to apoptosis are:
Include negative controls in the form of specific inhibitors of cathepsins, to make sure the measured activity is due only to cathepsin.
Use caspase inhibitors so that the activity due to various cysteine proteases can be distinguished. It must be remembered that there are general caspase inhibitors like z-FA-FMK which are capable of cathepsin inhibition as well, and therefore cathepsin inhibitors must also be used to provide controls.
During necrosis of cells, cathepsin activation occurs, which means it is not a specific indicator of apoptosis alone. For this reason the right type of controls must be used, along with the relevant cell lines, so that the study provides valid information.
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.
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