The effect of protease inhibitors and decreased temperature on the degradation of different classes of proteins in cultured hepatocytes

Abstract

Leupeptin, chymostatin and antipain inhibited the degradation of long-lived proteins in cultured rat hepatocytes by 20-30%, probably by inhibiting lysosomal proteases: (1) Leupeptin and chymostatin decreased to a similar extent the degradation of an exogenous protein 125I-asialo fetuin, a process known to occur within lysosomes. (2) In extracts of cells treated with leupeptin, cathepsin B activity was inhibited by 35-50%. (3) Leupeptin, chymostatin and antipain inhibited proteolysis by homogenates of liver lysosomes but not by the supernatant fraction. These agents, however, do not appear to rapidly permeate the membrane of isolated lysosomes. Leupeptin, chymostatin and antipain did not inhibit the breakdown of short-lived normal cell proteins, and ones containing amino acid analogs. Even when the amount of abnormal proteins was increased, such that it comprised a large fraction of cell protein, the degradation of these polypeptides was still very rapid and not affected by these inhibitors. The pathway for the degradation of short-lived cell proteins thus appears distinct from that responsible for degradation of long-lived cell proteins. In accord with this conclusion, reduction of the temperature of cultures inhibited the breakdown of long-lived proteins to a much greater extent than it affected the breakdown of short-lived ones. Treatment of cultured hepatocytes with glucagon, or deprivation for serum or amino acids stimulated the degradation of the more stable cell proteins but did not affect the breakdown of 125I-asialo-fetuin. Under these conditions leupeptin and chymostatin inhibited the breakdown of long-lived cell proteins to the same extent as in control cultures. Thus, lysosomal enzymes seem to play an important role in protein breakdown both in fed hepatocytes and in cells where proteolysis is accelerated.

Suppression of Cathepsins B and L Causes a Proliferation of Lysosomes and the Formation of Meganeurites in Hippocampus

“…These include an accumulation of lipofuscin (Ivy et al, 1984(Ivy et al, , 1989aNunomura and Miyagishi, 1993), a build-up of abnormally phosphorylated tau proteins in neuronal perikarya and dendrites (Ivy et al, 1989b;Takauchi and Miyoshi, 1995), increased levels of potentially amyloidogenic fragments (Hajimohammadreza et al, 1994), a decline in dopamine D 2 receptors in striatum (Shibata et al, 1992), and distended initial segments of axons (Cavanagh et al, 1993). Chloroquine is an acidotropic agent that disrupts protein degradation in lysosomes (Ohkuma, 1987), whereas the predominant action of leupeptin is to block cysteine proteases (Toyo-Oka et al, 1978;Neff et al, 1979). Accordingly, lysosomal cysteine proteases are the likely common targets of the drugs and, from the results cited above, it would appear that a subgroup of these enzymes is closely linked to well-established correlates of brain aging.…”

Renal proteinases and kidney hypertrophy in experimental diabetes

“…While we do not have direct experimental evidence, several observations suggest a close relationship. Inhibition of lysosomal cathepsins may reduce intracellular protein breakdown by up to 70 % [24][25][26], whereas protein degradation has been shown to be paralleled by enhanced cathepsin activity [27][28][29][30]. These findings support the view that lysosomal proteinases are related to intracellular protein turnover, such that reduced activity would favour protein accumulation.…”

Intracellular protein degradation: from a vague idea thru the lysosome and the ubiquitin–proteasome system and onto human diseases and drug targeting

“…The evolution of methods to monitor protein kinetics in cells together with the development of specific and general lysosomal inhibitors has resulted in the identification of different classes of cellular proteins, long-and short-lived, and the discovery of the differential effects of the inhibitors on these groups (see, e.g., Knowles and Ballard, 30 and Neff et al 31 ). An elegant experiment in this respect was carried out by Brian Poole and his co-workers in the Rockefeller University.…”