Effects of cysteine protease inhibitors on rabbit cathepsin D maturation
- Loyola Univ., Stritch School of Medicine, Maywood (USA)
To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation.
- OSTI ID:
- 7195065
- Journal Information:
- American Journal of Physiology; (USA), Vol. 257; ISSN 0002-9513
- Country of Publication:
- United States
- Language:
- English
Similar Records
Influence of cathepsin B and leupeptin on potentially lethal damage repair in mammalian cells
K-ras mutation promotes ionizing radiation-induced invasion and migration of lung cancer in part via the Cathepsin L/CUX1 pathway
Related Subjects
CATHEPSINS
MEMBRANE TRANSPORT
ENZYME INHIBITORS
BIOCHEMICAL REACTION KINETICS
PEPTIDE HYDROLASES
ENZYME ACTIVITY
CELL CULTURES
CYSTEINE
DOSE-RESPONSE RELATIONSHIPS
FIBROBLASTS
LEUCINE
LYSOSOMES
METHIONINE
MYOCARDIUM
PROTEOLYSIS
RABBITS
RADIOIMMUNOASSAY
SECRETION
SULFUR ISOTOPES
AMINO ACIDS
ANIMAL CELLS
ANIMALS
BIOASSAY
BODY
CARBOXYLIC ACIDS
CARDIOVASCULAR SYSTEM
CELL CONSTITUENTS
CHEMICAL REACTIONS
CONNECTIVE TISSUE CELLS
DECOMPOSITION
DIAGNOSTIC TECHNIQUES
DRUGS
ENZYMES
HEART
HYDROLASES
IMMUNOASSAY
IMMUNOLOGY
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIPOTROPIC FACTORS
MAMMALS
MUSCLES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANOIDS
ORGANS
RADIOASSAY
RADIOIMMUNODETECTION
RADIOIMMUNOLOGY
REACTION KINETICS
SH-PROTEINASES
SOMATIC CELLS
THIOLS
TRACER TECHNIQUES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques