Measuring Cysteine Cathepsin Activity to Detect Lysosomal Membrane Permeabilization.

During lysosomal membrane permeabilization (LMP), lysosomal lumenal contents can be released into the cytosol. Small molecules are more likely to be released, and cysteine cathepsins, with mature forms possessing a mass of 25-30 kDa, are among the smallest lumenal lysosomal enzymes. In addition, specific substrates for cysteine cathepsins are available to investigators, and therefore the measurement of the cathepsin activity as a hallmark of LMP works well. Here, we present a protocol for measuring the activity of these enzymes after selective plasma membrane permeabilization with a low concentration of digitonin and after total cell membrane lysis with a high concentration of digitonin. A fluorogenic substrate can be added either directly to the well with lysed cells to show LMP or to the cell-free extract to show that the lysosomal membrane has been sufficiently destabilized to allow the translocation of lysosomal enzymes. Although the content of lysosomal cysteine cathepsins differs between cell lines, this method has general applicability, is sensitive, and has high throughput. The presented protocol shows how to measure cysteine cathepsin activity in the presence of lysed cells and also in cell-free extracts. Depending on the aim of the study, one or both types of measurements can be performed.

Lysosomal pathways to cell death and their therapeutic applications.

Lysosomes are the major cell digestive organelles that were discovered over 50 years ago. They contain a number of hydrolases that help them to degrade intracellular and extracellular material delivered. Among the hydrolases, the cathepsins, a group of proteases enclosed in the lysosomes, have a major role. About a decade ago, the cathepsins were found to participate in apoptosis. Following their release into the cytosol, they cleave Bid and degrade antiapoptotic Bcl-2 proteins, thereby triggering the mitochondrial pathway of apoptosis, with the lysosomal membrane permeabilization being the critical step in this pathway. Lysosomal dysfunction is linked with several diseases, including cancer and neurodegenerative disorders, thereby providing a potential for therapeutic applications. In this review lysosomes and lysosomal proteases involvement in apoptosis and their possible pharmaceutical targeting are discussed.