Non-lysosomal Protein Degradation Research Articles

Distinction between major chloroquine-inhibitable and adrenergic-responsive pathways of protein degradation and their relation to tissue ATP content in the Langendorff isolated perfused rat heart.

In the Langendorff isolated perfused rat heart, 36% of total basal protein degradation was inhibited by the lysosomal inhibitor chloroquine (30 microM), after elimination of rapid turnover proteins during a 3 h preliminary degradation period. Prior inhibition of degradation with chloroquine was additive to the 30% inhibition caused by simultaneous infusion of 50-200 nM-isoprenaline. This additivity suggests that the adrenergic-controlled process is independent of the lysosomal degradative pathway. After discontinuation of drug infusions, the isoprenaline-inhibited degradation rate returned to the previous baseline; however, the chloroquine-inhibited degradation rate transiently exceeded the previous baseline. NaN3 (0.3 mM) caused a decrease of left-ventricular myocardial ATP content of approx. 60% at 14 min and extreme impairment of contractile function; however, the total lysosomal and non-lysosomal protein degradation was not changed at this time. Conversely, left-ventricular tissue ATP content was not changed during proteolytic inhibition by 10 nM-isoprenaline or 10 microM-chloroquine at 14 min. The results indicate that depletion of myocardial energy stores in this preparation is neither necessary nor sufficient to cause inhibition of the total of lysosomal and non-lysosomal protein degradation.

Temperature dependence of protein degradation, autophagic sequestration and mitochondrial sugar uptake in rat hepatocytes

Lysosomal (propylamine-sensitive) protein degradation as well as the energy-dependent (chymostatin-sensitive) part of the non-lysosomal protein degradation was found to be strongly affected by temperature in isolated rat hepatocytes, the activation energy ( E a) being about 25 kcal/mol for both processes. In contrast, the energy-independent (chymostatin-resistant) part of the non-lysosomal degradation had an E a of approx. 10 kcal/mol only. Sequestration of electroinjected [ 14C]sucrose into sedimentable organelles showed a pronounced temperature dependence. By means of digitonin extraction it was possible to distinguish between a moderately temperature-sensitive mitochondrial sugar uptake ( E a approx. 12 kcal/mol) and a strongly temperature-dependent autophagic sequestration ( E a approx. 22 kcal/mol). There was no significant autophagic sequestration below 20°C. The sequestration process is more temperature-sensitive than, for example, the early steps of endocytosis, and is likely to represent the major controlling step in the overall autophagic-lysosomal pathway.

Differential effects of proteinase inhibitors and amines on the lysosomal and non-lysosomal pathways of protein degradation in isolated rat hepatocytes

Ammonia, which like other lysosomotropic amines inhibits protein degradation in isolated rat hepatocytes by 70–80%, was utilized as a diagnostic tool to distinguish between the relative effects of various proteinase inhibitors on the lysosomal and non-lysosomal pathways of intracellular protein degradation. Leupeptin was found to inhibit lysosomal protein degradation by 80–85%, and non-lysosomal degradation by about 15%. Antipain had a similar, but somewhat weaker effect. Pepstain, bestatin and aprotinin (Traysylol) produced minor inhibitory effects (possibly on both degradation, pathways), whereas bacitracin and soybean trypsin inhibitor wre ineffective. Chymostatin inhibited lysosomal protein degradation by about 45%, whereas the non-lysosomal pathway was inhibited by more than 50%. Chymostatin was unique among the inhibitors tested in causing such a pronounced effect on non-lysosomal protein degradation, and appeared to selectively inhibit the energy-dependent portion of this pathway. The effects of the various inhibitors were additive to the extent expected on the basis of their kwown actions on lysosomal and non-lysosomal protein degradation. Thus, a combination of methylamine, leupeptine and chymostatin inhibited overall protein degradation by about 90%, resulting in a substantial improvement of the cellular nitrogen balance. The degradation inhibitors caused a partial inhibition of protein synthesis, apparently mainly by shutting down the supply of amino acids from the lysosome. The inhibitory effects of leupeptin and antipain were completely reversed by amino acid addition, whereas some inhibition remained in the case of chymostatin and the lysosomotropic amines, possibly reflecting a certain nonspecific toxicity.