Metalloendoprotease inhibitors block protein synthesis, intracellular transport, and endocytosis in hepatoma cells.
Fusion of membrane vesicles has been implicated in many intracellular processes including the transport of proteins destined for secretion or storage. Vesicular transport coupled with membrane fusion has been demonstrated for rough endoplasmic reticulum to Golgi and Golgi to plasma membrane transport as well as receptor mediated endocytosis and receptor recycling. Recent studies with inhibitors suggest that metalloendoproteases may mediate a wide variety of intracellular fusion events. Thus, in order to examine the potential role of metalloendoproteases in both transport/secretion and endocytosis/recycling we have used selected dipeptide substrates to probe these processes in human HepG2 cells. Using pulse-chase labeling, immunoprecipitation, and polyacrylamide gel electrophoresis we show that transport and secretion of newly synthesized proteins along the exocytotic route were completely inhibited by substrate dipeptides (e.g. Cbz-Gly-Phe-amide, where Cbz is benzyloxycarbonyl) but not by irrelevant dipeptides (e.g. Cbz-Gly-Gly-amide). The effect was rapid, reversible, and specific. The secretory pathway was blocked between the rough endoplasmic reticulum and Golgi as well as Golgi and plasma membrane as judged by the status of N-glycosylation intermediates. In addition, these inhibitors specifically inhibited protein synthesis without alterations in cellular ATP concentrations. However, cell-free amino acid incorporation was not inhibited. Receptor-mediated uptake of asialoglycoproteins was specifically and reversibly inhibited by dipeptide substrates. This effect appears to be secondary to inhibition of recycling as neither ligand binding nor internalization were affected. Thus the present observations suggest that metalloendoprotease activity may be involved in the regulation of multiple intracellular pathways perhaps at the level of vesicular fusion events.
Strous
G J
GJ
Laboratory for Cell Biology, Medical School, University of Utrecht, The Netherlands.
van Kerkhof
P
P
Dekker
J
J
Schwartz
A L
AL
D001692:Biological Transport / Q000187:drug effects; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002460:Cell Line; D004151:Dipeptides / Q000494:pharmacology*; D004705:Endocytosis / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D009363:Neoplasm Proteins / Q000096:biosynthesis; D011500:Protein Synthesis Inhibitors; D013329:Structure-Activity Relationship
D004151:Dipeptides; D009363:Neoplasm Proteins; D011500:Protein Synthesis Inhibitors; D008666:Metalloendopeptidases