alpha-chymotrypsin and epoxomicin

A growing body of evidence suggests that α-synuclein accumulation may play an important role in the pathogenesis of Parkinson's disease. The aim of this study was to investigate the roles of the proteasome and autophagy pathways in the clearance of wild-type and mutant α-synuclein in PC12 cells.. PC12 cells overexpressing either wild-type or A30P mutant α-synuclein were treated with the proteasome inhibitor epoxomicin, the macroautophagy inhibitor 3-MA and the macroautophagy activator rapamycin alone or in combination. The cell viability was assessed using MTT assay. Immunofluorescence and Western blot analysis were used to detect the level of α-synuclein, LAMP-2A, E1 activase, and E2 ligase in the cells. Chymotrypsin-like proteasomal activity was measured using a commercial kit.. When the proteasome and macroautophagy in the wild-type and mutant cells were inhibited with epoxomicin and 3-MA, respectively, the cell viability was significantly decreased, and the α-synuclein level was increased. Both epoxomicin and 3-MA activated the chaperone-mediated autophagy (CMA) by increasing the level of the CMA-limiting enzyme LAMP-2A. Furthermore, 3-MA or epoxomicin significantly decreased chymotrypsin-like proteasomal activity. 3-MA or epoxomicin did not change E1 activase expression in either mutant or wild-type cells, but increased E2 ligase expression, especially when used together. Macroautophagy inducer rapamycin increased the cell viability and reduced epoxomicin-induced α-synuclein accumulation. Interestingly, CMA was also activated by rapamycin.. Our results demonstrate the existence of complex crosstalk between different forms of autophagy and between autophagy and the proteasome pathway in the clearance of α-synuclein in PC12 cells.

Topics: Adenine; alpha-Synuclein; Animals; Autophagy; Cell Survival; Chymotrypsin; Humans; Oligopeptides; Parkinson Disease; PC12 Cells; Point Mutation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats

The ubiquitin-proteasome system controls a variety of essential intracellular processes through directed protein turnover. The invertebrate proteasome has recently gained increasing interest with respect to central physiological processes and pathways in different taxa. A pitfall in proteasome activity assays, represented by the trypsin-like, chymotrypsin-like or caspase-like site, lies in the fact that most commonly used experimental substrates are susceptible to degradation by non-proteasomal proteolytic enzymes, which can lead to erroneous interpretation of activity data obtained. Through the use of a proteasome-specific inhibitor, epoxomicin, we showed that the shares of proteasomal and non-proteasomal activities in the degradation of a model polypeptide substrate for chymotrypsin-like activity vary considerably between invertebrate taxa. Crustacean muscle tissue and hemocytes showed almost exclusively proteasomal activity. In yeast, approximately 90% of total proteolytic activity can be attributed to the proteasome. In contrast, proteasomal activity comprises only 20-60% of the total proteolytic activity in bivalve tissues. These results reveal that, without verification of the shares of proteasomal and non-proteasomal activities in crude extracts through the use of highly specific inhibitors, common proteasomal enzyme assays should be used and interpreted with caution.

Topics: Animals; Bivalvia; Chymotrypsin; Crustacea; Enzyme Assays; Hemocytes; Muscles; Oligopeptides; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Proteolysis

The ubiquitin proteasome-proteolytic pathway has emerged as one of the most significant pathways in modulating protein homeostasis under both normal and disease states. The use of proteasome inhibitors (PI) has played a pivotal role in understanding protein turn over. The main objective of this work was to develop a comprehensive, fast, and reliable, yet simple in vitro assay that would allow for the identification and characterization of a wide range of PIs. The assays consist of a 96-well plate high throughput (HTP) method to assess proteasome activity in Hs578T breast cancer cell extracts, purified 20S proteasome, using a fluorogenic substrate, Suc-leu-leu-val-tyr-7-AMC, specific to the chymotrypsin-like enzymatic activity of the proteasome. We showed that the chymotrypsin-like activity of the proteasome was inhibited in the two in vitro systems, albeit to different degrees. The assay system also includes two cell-based assays consisting of a vector expressing a fusion protein of green fluorescent protein (gfp) and Mouse Ornithine Decarboxylase (MODC) in Zs578T (parental Hs578T carrying the vector that expresses the fusion protein). In the cell-based assay analyses (qualitatively by microscopy and quantitatively by flow cytometry), treatment of Zs578T with PIs prevented the degradation of MODC, accumulated gfp, indicative of increased proteasome inhibition. Because no single assay represents a definitive proof of proteasome inhibitory activity, combined, these assays should serve as a comprehensive benchmark for the identification and partial characterization of novel inhibitors. In summary, the four-step assay protocol can easily be adapted into a high throughput format to rapidly screen unknown inhibitors.

Topics: Acetylcysteine; Animals; Biological Assay; Boronic Acids; Bortezomib; Cell Extracts; Cells, Cultured; Chymotrypsin; High-Throughput Screening Assays; Inhibitory Concentration 50; Leupeptins; Mice; Models, Biological; Oligopeptides; Ornithine Decarboxylase; Protease Inhibitors; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Pyrazines; Recombinant Fusion Proteins; Time Factors

We evaluated whether the proteasomal chymotrypsin-like (ChT-L) activity is increased in plasma of patients with acute lymphoblastic (ALL), acute myeloblastic (AML) and chronic lymphocytic (CLL) leukemias.. The activity was assayed using the fluorogenic peptide substrate in the presence of an artificial activator sodium dodecyl sulfate (SDS) in the plasma of healthy donors (n=15) and ALL (n=15), AML (n=28) and CLL (n=22) patients.. The activity was significantly (P<0.001) higher in the plasma of ALL and AML patients at the diagnosis than in healthy subjects and decreased after therapy or remained unchanged or rose during relapse. By contrast, in CLL patients at the diagnosis, the activity did not differ significantly from the healthy controls. In each group, the activity positively correlated with the serum lactic dehydrogenase activity.. Plasma proteasome ChT-L activity can be a useful bio-marker for patients with acute leukemia at the blast stage.

Topics: Adolescent; Adult; Aged; Case-Control Studies; Chymotrypsin; Female; Humans; Hydrolysis; L-Lactate Dehydrogenase; Leukemia; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Myeloid, Acute; Male; Middle Aged; Oligopeptides; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Subunits; Sodium Dodecyl Sulfate