Internalisation of Short-Chain Isoprenyl Diphosphates by Chromaffin Cells from Bovine Adrenal Medulla

Dessel, G. Van; Busser, H. de; Lagrou, A.

doi:10.1006/bbrc.1997.7230

Cited by 3 publications

(4 citation statements)

References 7 publications

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“…Temperature-independent membrane permeability of FPP and GGPP has been previously demonstrated . The details of the process are currently unclear, as it is unknown whether the isoprenoids cross the membrane in the form of pyrophosphates or alcohols.…”

Section: Resultsmentioning

confidence: 99%

“…Temperature-independent membrane permeability of FPP and GGPP has been previously demonstrated. 50 The details of the process are currently unclear, as it is unknown whether the isoprenoids cross the membrane in the form of pyrophosphates or alcohols. The latter are more likely to be able to cross the membrane because they are much more hydrophobic than the prenyl pyrophosphates.…”

Section: Fluorescent Substrates Of Protein Prenyltransferasesmentioning

confidence: 99%

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Identification and Specificity Profiling of Protein Prenyltransferase Inhibitors Using New Fluorescent Phosphoisoprenoids

et al. 2006

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Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.

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Section: Resultsmentioning

confidence: 99%

Section: Fluorescent Substrates Of Protein Prenyltransferasesmentioning

confidence: 99%

Identification and Specificity Profiling of Protein Prenyltransferase Inhibitors Using New Fluorescent Phosphoisoprenoids

et al. 2006

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“…The mechanism by which the charged pyrophosphates enter the cell is not well-understood. Previous studies have shown that the uptake of radiolabeled FPP or GGPP is not significantly affected by the temperature of the incubation medium or by metabolic poisons, suggesting that the process is energy-independent. It should be noted that in plants, there appears to be a unidirectional transport system in the chloroplast envelope membrane which mediates the export of isoprenoid pyrophosphates (IPP, GPP > FPP, DMAPP) from the chloroplast to the cytosol …”

Section: Resultsmentioning

confidence: 94%

“…However, it is known that under conditions of mevalonate depletion induced by an HMG-CoA reductase inhibitor, addition of FPP or GGPP can restore protein farnesylation or geranylgeranylation, respectively . In addition, radiolabeled FPP or GGPP can become incorporated into isoprenylated proteins . Such experiments can only be successful if the isoprenoid pyrophosphates penetrate the cell membrane at a level sufficient to restore a significant internal concentration.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and Activity of Fluorescent Isoprenoid Pyrophosphate Analogues

et al. 2004

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New fluorescent analogues of farnesol and geranylgeraniol have been prepared and then converted to the corresponding pyrophosphates. These analogues incorporate anthranylate or dansyl-like groups anchored to the terpenoid skeleton through amine bonds that would be expected to be relatively stable to metabolism. After addition of the alcohols or the pyrophosphates to the culture medium, their fluorescence is readily observed inside a human-derived leukemia cell line. Enzyme assays have revealed that the farnesyl pyrophosphate analogue is an inhibitor of FTase, while the corresponding alcohol is not. These results, together with Western blot analyses of cell lysates, indicate that the farnesyl pyrophosphate analogue penetrates the cells as an intact pyrophosphate and that it does so at a biologically relevant concentration.

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