Regulation of the Activity of Lysosomal Cysteine Proteinases by pH-Induced Inactivation and/or Endogenous Protein Inhibitors, Cystatins

Abstract

The kinetics of pH-induced inactivation of human cathepsins B and L was studied by conventional and stopped-flow methods. The inactivation of both enzymes was found to be an irreversible, first-order process. The inactivation rate constants increased exponentially with pH for both enzymes. From log kinac vs pH plots, 3.0 and 1.7 protons were calculated to be desorbed for pH-induced inactivation of cathepsins L and B. Cathepsin B was thus substantially more stable than cathepsin L (approximately 15-fold at pH 7.0 and 37 degrees C). Cathepsin B was efficiently inhibited by cystatin C at pH 7.4, whereas the inhibition by stefin B and high molecular weight kininogen was only moderate. In contrast, cathepsin L was efficiently inhibited by both chicken cystatin and stefin B at this pH kass approximately 3.3 x 10(7) m-1 s-1).

Lysosomes in cell death

“…One of the most controversial issues regarding the role of lysosomal proteases outside the lysosomes is their actual ability to function at neutral pH. A series of elegant studies showed that, although lysosomal enzymes have an activity optimum at acidic pH, lysosomal cysteine proteases are stable and active at neutral pH for a time that ranges from a few minutes to an hour or more, confirming their destructive potential in cellular compartments other than the lysosomes (Turk et al, 1993(Turk et al, , 1995(Turk et al, , 2000. Cathepsin B, which is one of the most stable proteases at physiologic pH, is essential in different models of apoptosis, including bile acid-induced hepatocyte apoptosis (Roberts et al, 1997;Jones et al, 1998;Faubion et al, 1999;Canbay et al, 2003), TNF-ainduced apoptosis of primary hepatocytes and tumor cells (Guicciardi et al, 2000(Guicciardi et al, , 2001Foghsgaard et al, 2001), neuronal apoptosis in Unverricht-Lundborg progressive myoclonus epilepsy (EPM1) (Houseweart et al, 2003a) and after brain ischemia (Yamashima et al, 1998;Tsuchiya et al, 1999), and PC12 cell apoptosis after serum deprivation (Shibata et al, 1998).…”

Cathepsin B Degrades Amyloid-β in Mice Expressing Wild-type Human Amyloid Precursor Protein

“…CysC Reduction Lowers A␤ Levels without Affecting hAPP WT Processing-CatB activity is blocked by its endogenous inhibitor, CysC (cst3) (17,18). To further explore the effects of CatB activity on A␤ and hAPP WT processing, we crossed hAPP WT mice with cst3 Ϫ/Ϫ mice.…”

Lysosomes as “Suicide Bags” in Cell Death: Myth or Reality?

“…There are several possible reasons for this, the most likely being instability of cysteine cathepsins and almost zero activity of cathepsin D at the neutral pH of the cytosol. Whereas cysteine cathepsins lose their activity due to irreversible unfolding (35), cathepsin D activity is lost reversibly due to deprotonation of both active-site Asp residues. Additional reasons probably lie with the substrates: (i) their structures are probably more compact at neutral pH and thereby less accessible to proteolysis, and (ii) it is likely that some proteins are inaccessible to proteolysis due to their involvement in complex formation and/or membrane attachment.…”

“…Cathepsins therefore pose a permanent latent threat to cells in cases where there is significant damage to lysosomes. Control of this threat is maintained by their intracellular inhibitors, stefins and serpins, which serve as cellular guardians, playing a more efficient role than just carrying out simple pH-induced, irreversible inactivation (35,53). This activity became even more evident when the major intracellular inhibitor of the cathepsins, stefin B, was ablated, and stefin B-deficient mice were observed to spontaneously develop cerebellar apoptosis (42).…”