CysC enhances total lysosomal-dependent protein degradation in serum-deprived neuronal cells.
A. Effect of increasing concentrations of CysC on total rates of protein degradation. N2a cells were labeled for 2 days with [3H]-leucine. After extensive washing, cells were incubated in serum-containing or serum-free media. Removal of serum maximally activates lysosomal degradation. The cells maintained in serum-free media were supplemented or not with increasing concentrations of CysC as labeled. The rate of total protein degradation at the indicated times was calculated as the percentage of total radiolabeled protein transformed into soluble amino acids. B. Effect of inhibition of lysosomal proteolysis on the CysC-induced increase in protein degradation. N2a cells were labeled as in A and then maintained in serum-free media and supplemented or not with CysC. Where indicated 20 mM NH4Cl and 100 µM leupeptin were added to inhibit lysosomal proteolysis. Protein degradation was calculated as in A. C. Effect of CysC on macroautophagy-dependent proteolysis. N2a cells labeled as in A and maintained in serum-free media were supplemented or not with CysC. Half of the cells were treated with 10 mM 3MA to inhibit macroautophagy. The percentage of lysosomal degradation that results from autophagic degradation (3MA sensitive), in the presence or absence of CysC was calculated. Values are mean and SED of triplicate wells in 3–4 different experiments. One way ANOVA for differences between CysC treated and untreated samples were significant for *p = 0.05; **p = 0.001 and between control and ammonium chloride treated samples were significant for +p = 0.01.