All tags Apoptosis Role of calpains and cathepsins in apoptosis

Role of calpains and cathepsins in apoptosis

Non-caspase proteases play an important role in cell death.

Inhibition of caspases only results in a delay and not a complete inhibition of apoptosis, highlighting
the essential role of non-caspase proteases such as calpains and cathepsins

​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

​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:

  • Ensure that there is no contamination from protease-rich lysosomes or other organelles that would give a false positive. Consider using inhibitors to lysosomal proteases (cathepsins) to limit non-specific activity.
  • Similarly, it is a good idea to use generic caspase inhibitors to be able to differentiate between the different cysteine proteases.
  • Treat cells with calpain-specific inhibitors as negative controls to ensure the activity you are measuring is calpain dependent.
  • Calpain is likely to be active at some level in the cell, so use the appropriate background controls (untreated cells) to provide a baseline for calpain activation.
  • Calpain activity itself is not an indication of apoptosis, as calpain has other functions and is activated during necrosis. Make sure that you are using appropriate controls and look at other relevant apoptotic parameters.
ab65308 calpain activity assay

​​​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

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:

  • Use specific cathepsin inhibitors as negative controls to ensure the activity you are measuring is cathepsin-specific.
  • Use caspase inhibitors in your study to be able to differentiate between the activities of the different cysteine proteases. Unfortunately, some general caspase inhibitors such as z-FA-FMK can also inhibit cathepsin activity so ensure that you include cathepsin inhibitors as controls.
  • Cathepsin are also activated during necrosis, and cathepsin activation is not a unique indication of apoptosis. Ensure that you are using appropriate controls and the appropriate cell line to give content to your study.
ab65300 cathepsin b activity assay kit

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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