A potent oligosaccharyl transferase inhibitor that crosses the intracellular endoplasmic reticulum membrane
| Autor: | Paula Denney Eason, Barbara Imperiali |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Glycosylation
Cell Membrane Permeability Stereochemistry Molecular Sequence Data Biological Transport Active Peptide Saccharomyces cerevisiae Biology Endoplasmic Reticulum Biochemistry Peptides Cyclic Substrate Specificity Diffusion Fungal Proteins chemistry.chemical_compound Mice Transferases Transferase Animals Asparagine Amino Acid Sequence ATP Binding Cassette Transporter Subfamily B Member 2 Enzyme Inhibitors chemistry.chemical_classification Endoplasmic reticulum membrane Endoplasmic reticulum Membrane Proteins Intracellular Membranes Mice Inbred C57BL Enzyme chemistry Hexosyltransferases ATP-Binding Cassette Transporters Female Intracellular |
| Zdroj: | Biochemistry. 38(17) |
| ISSN: | 0006-2960 |
| Popis: | Recent work has resulted in the development of potent inhibitors of oligosaccharyl transferase (OT), the enzyme that catalyzes the cotranslational glycosylation of asparagine [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637; Kellenberger, C., Hendrickson, T. L., and Imperiali, B. (1997) Biochemistry 36, 12554-12559]. However, no specific OT inhibitors that function in the cellular environment have yet been reported. The peptide cyclo(hex-Amb-Cys)-Thr-Val-Thr-Nph-NH2 was previously shown to exhibit nanomolar inhibition (Ki = 37 nM) through slow tight binding kinetics [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637]. Included herein is the redesign of this prototype inhibitor for achieving both passive and active translocation into model membrane systems representing the endoplasmic reticulum (ER). The strategy for passive transport involved the incorporation of a membrane permeable import function previously shown to carry various peptides across the outer as well as the interior cellular membranes [Rojas, M., Donahue, J. P., Tan, Z., and Lin, Y.-Z. (1998) Nat. Biotechnol. 16, 370-375]. Assessment of function in intact ER membranes revealed that the inhibitor targeted toward passive diffusion demonstrated concentration-dependent inhibition of two different glycosylation substrates. Thus, this modified inhibitor achieved potent inhibition of glycosylation after being successfully transported through the ER membrane. In the active translocation approach, the lead OT inhibitor and a corresponding substrate were redesigned to include features recognized by the transporter associated with antigen processing (TAP). This protein translocates peptides into the lumen of the ER [Heemels, M.-T., Schumacher, T. N. M., Wonigeit, K., and Ploegh, H. L. (1993) Science 262, 2059-2063]. However, although acceptance of the cyclized substrate by the TAP receptor was demonstrated via efficient transport and glycosylation, the modified inhibitor was not translocated by TAP machinery, and therefore, active translocation was achieved for the modified substrate only. Both of these ER transport methods afforded redesigned OT inhibitors that retained their inhibitor properties in vitro, regardless of the extensions to the carboxy-terminus of the root inhibitor. The above family of redesigned inhibitors provides a template for generating a transcellular pathway and represents the first step toward OT inhibition in intact cells. |
| Databáze: | OpenAIRE |
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