WHY ISOPRENOIDS?

According to Sacchettini & Poulter¹ over 23,000 individual isoprenoid compounds have been characterized. These include: cholesterol, bile acids, steroid hormones, dolichol, ubiquinone, prenylated proteins and a wide variety of plant terpenoids. (See: http://www.wdv.com/CellWorld/Biochemistry/Isoprenoids/ for a schematic of the isoprenoid pathway in mammals, and http://www.genome.jp/kegg/pathway/hsa/hsa00900.html for a map of terpenoid biosynthesis). In order to study the metabolism of these compounds in vitro and in vivo, researchers need a ready, inexpensive supply of precursors, the isoprenoid pyrophosphates (also known as isoprenoid diphosphates). These include dimethylallyl pyrophosphate, isopentenyl pyrophosphate, geranyl pyrophosphate, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. Studies over the last decade^2-5 indicate that, contrary to previously held beliefs, isoprenoid pyrophosphates enter whole cells. These findings allow investigations into the ability of various isoprenoid pyrophosphates to reverse the effects of statins on HMG-CoA reductase. Is the effect one observes of a statin due to an inhibition of protein geranylgeranylation? Simple: add back GGPP and see if it reverses the effect. Farnesyl pyrophosphate is the control, since in the absence of IPP, GGPP cannot be generated. Confirmation of the experimental results can be achieved using farnesyl and geranylgeranyl protein transferase inhibitors.

The Schroepfer Medal of 2008 went to Michel Rohmer, who discovered the non-mevalonate pathway of isoprenoid metabolism⁶ which occurs in many plants and bacteria. The primary inhibitor of this pathway is fosmidomycin. Researchers who wish to examine the effects of fosmidomycin on this pathway may want to employ the isoprenoid pyrophosphates in the same manner as used for studies of statins in the classical pathway, as discussed above.

Studies indicate bisphosphonates, widely used in the treatment of osteoporosis, inhibit at FPP synthase^7-9. As with statins, isoprenoid pyrophosphates can be used to examine the effects of this inhibition on various cellular processes.

It is often desirable to determine the levels of isoprenoid pyrophosphates under conditions in which the mevalonate pathway is greatly altered (hormones, statins, cholesterol feeding). A new method has just been published that allows direct assay of FPP and GGPP in cultured cells¹⁰, and mammalian tissue¹¹ indirect assays of IPP have been published¹².

Studies indicate that the level of protein prenylation may be regulated¹³. It may be possible to change the level of prenylation by administering isoprenoid pyrophosphates to cells or even whole animals.

A certain class of T-cells has been shown to be activated by isoprenoid pyrophosphates¹⁴. One can now examine the mechanism of this activation by IPP and other isoprenoid pyrophosphates.

Large quantities of terpenoids, sesquiterpenoids and diterpenoids can be synthesized using cloned enzymes and isoprenoid pyrophosphate substrates.

With our inexpensive isoprenoid pyrophosphates, you can now do these experiments!

1. Sacchettini, James C.; Poulter, C. Dale (1997) Creating Isoprenoid Diversity. Science 277(5333) 1788-1789.

2. Danesi R, McLellan CA, Myers CE. 1995 Specific labeling of isoprenylated proteins: application to study inhibitors of the post-translational farnesylation and geranylgeranylation. Biochem Biophys Res Commun. 206(2):637-43.

3. Tanaka T, Tatsuno I, Uchida D, Moroo I, Morio H, Nakamura S, Noguchi Y, Yasuda T, Kitagawa M, Saito Y, Hirai A. 2000. Geranylgeranyl pyrophosphate, an isoprenoid of mevalonate cascade, is a critical compound for rat primary cultured cortical neurons to protect the cell death induced by 3-hydroxy-3-methylglutaryl-CoA reductase inhibition. J Neurosci. 20(8):2852-9.

4. Cicha, I., et al. Monitoring the cellular effects of HMG-CoA reductase inhibitors in vitro and ex vivo. ArteriosclerThromb Vasc Biol. 2004 Nov;24(11):2046-5

5. Mandey, SH, et al. A role for geranylgeranylation in interleukin-1beta secretion. ArthritisRheum. 2006 Nov;54(11):3690-5)

6. Rohmer M, Knani M, Simonin P, Sutter B, Sahm H. Isoprenoid biosynthesis in bacteria: a novel pathway for the early steps leading to isopentenyl diphosphate. Biochem J. 1993 Oct 15;295

7. Keller, RK and Fliesler, SJ (1999) Mechanism of aminobisphosphonate action: characterization of alendronate inhibition of the isoprenoid pathway. Biochem Biophys Res Commun. 1999 Dec 20;266(2):560-3.

8. van Beek, E. et al. (1999) Nitrogen-Containing Bisphosphonates Inhibit Isopentenyl Pyrophosphate Isomerase/Farnesyl Pyrophosphate Synthase Activity with Relative Potencies Corresponding to Their Antiresorptive Potencies in Vitro and in Vivo. Biochem. Biophys. Res. Commun. 255, 491-494.

9. Bergstrom JD, et al. (2000) Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch Biochem Biophys. 373(1):231-41.

10. Tong H, Holstein SA, Hohl RJ. 2005 Simultaneous determination of farnesyl and geranylgeranyl pyrophosphate levels in cultured cells. Anal Biochem. 336(1):51-9.

11. Tong H, Wiemer AJ, Neighbors JD, Hohl RJ. 2008.Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue. Anal Biochem. Apr 15

12. Keller, RK. (1996) Squalene synthase inhibition alters metabolism of nonsterols in rat liver. Biochim Biophys Acta. 1303(3):169-79.

13. David Vicent, Eleftheria Maratos-Flier, and C. Ronald Kahn (2000) The branch point enzyme of the mevalonate pathway for protein prenylation is overexpressed in the ob/ob mouse and induced by adipogenesis. Mol Cell Biol. Mar;20(6):2158-66.

14. Mori, L.; De Libero, G. (1995) Human V 9-V 2 cells are stimulated in a cross-reactive fashion by a variety of phosphorylated metabolites. Eur. J. Immunol. 25, 2052-2058