Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyltransferase that specifically prevents Rab prenylation in osteoclasts and macrophages

Fraser Coxon, M. H. Helfrich, B. Larijani, M. Muzylak, James Edward Dunford, D. Marshall, Alastair D McKinnon, S. A. Nesbitt, M. A. Horton, M. C. seabra, F. H. Ebetino, Michael John Rogers

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Abstract

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak antiresorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass similar to 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [H-3]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [C-14]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.

Original languageEnglish
Pages (from-to)48213-48222
Number of pages9
JournalThe Journal of Biological Chemistry
Volume276
Issue numberDec 21
DOIs
Publication statusPublished - 2001

Keywords

  • NITROGEN-CONTAINING BISPHOSPHONATES
  • FARNESYL DIPHOSPHATE SYNTHASE
  • BONE-RESORPTION
  • PROTEIN PRENYLATION
  • IN-VITRO
  • MEVALONATE PATHWAY
  • SQUALENE SYNTHASE
  • MECHANISM
  • GTPASES
  • GROWTH

Cite this

Coxon, F., Helfrich, M. H., Larijani, B., Muzylak, M., Dunford, J. E., Marshall, D., McKinnon, A. D., Nesbitt, S. A., Horton, M. A., seabra, M. C., Ebetino, F. H., & Rogers, M. J. (2001). Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyltransferase that specifically prevents Rab prenylation in osteoclasts and macrophages. The Journal of Biological Chemistry, 276(Dec 21), 48213-48222. https://doi.org/10.1074/jbc.M106473200