Inhibition of translational initiation by metalloendoprotease antagonists. Evidence for involvement of sequestered Ca2+ stores.
Synthetic oligopeptide inhibitors of metalloendoprotease activity have been shown to block membrane fusion events, to slow transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi, and to perturb Ca2+ homeostasis. Effects of such agents on translational activity, which requires Ca2+ sequestered putatively within the ER, were examined in this study. Cbz-Gly-Phe-NH2 (where Cbz is benzyloxycarbonyl) provoked rapid inhibition of amino acid incorporation into a broad spectrum of proteins in GH3 pituitary, C6 glial, and Neuro-2a cells but not in reticulocytes, which lack ER. Polysome accumulation and incorporation were reduced concurrently, indicating that the dipeptide acted to slow translational initiation. Inhibitions were largest at low extracellular Ca2+, were reversed by increasing extracellular Ca2+, were comparable to those achieved in the presence of EGTA or Ca2+ ionophores, and were observed with assorted metalloendoprotease antagonists but not with leupeptin. At concentrations inhibitory to protein synthesis Cbz-Gly-Phe-NH2 mobilized cell-associated 45Ca, lowered cytosolic free Ca2+, and did not generate inositol phosphates. Cells treated for 3-4 h with Cbz-Gly-Phe-NH2 reacquired the ability to synthesize proteins at nearly normal rates; a phorbol ester or cAMP-elevating agent was necessary for such recovery in GH3, but not C6 or Neuro-2a, cells. GRP78, which may function in the folding and assembly of secretory proteins and in translational accommodation to agents that deplete sequestered Ca2+ stores, was induced during such treatments. Accumulation of GRP78 mRNA in treated preparations was reduced as extracellular Ca2+ was increased. Extended exposure to dipeptide followed by brief recovery in its absence rendered protein synthesis resistant to inhibition by Ca2+ ionophore. It is concluded that metalloendoprotease antagonists suppress translational initiation as a consequence of their capacity to mobilize sequestered Ca2+ stores.
Brostrom
M A
MA
Department of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854.
Prostko
C R
CR
Gmitter-Yellen
D
D
Grandison
L J
LJ
Kuznetsov
G
G
Wong
W L
WL
Brostrom
C O
CO
D000595:Amino Acid Sequence; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002352:Carrier Proteins / Q000235:genetics; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology*; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors*; D018832:Molecular Chaperones; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D010442:Peptide Chain Initiation, Translational / Q000187:drug effects*; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D002352:Carrier Proteins; D004151:Dipeptides; D000091342:Endoplasmic Reticulum Chaperone BiP; D006360:Heat-Shock Proteins; D007143:Immunoglobulin Heavy Chains; D007295:Inositol Phosphates; D007976:Leupeptins; D018832:Molecular Chaperones; D009842:Oligopeptides; D012333:RNA, Messenger; D003609:Dactinomycin; D015240:Phorbol 12,13-Dibutyrate; D000001:Calcimycin; D003513:Cycloheximide; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases; D007930:Leucine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium