Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the prenylation and membrane localization of Rab proteins in osteoclasts in vitro and in vivo
Introduction
Protein prenylation is a post-translational modification involving the transfer of either a farnesyl or geranylgeranyl isoprenoid lipid to the c-termini of specific target proteins, chiefly small GTPases of the Ras, Rho, and Rab families. This modification is critical for the function of these proteins, since it is required for membrane association and for specific protein–protein interactions [1]. The process of prenylation is carried out by one of three distinct protein:prenyl transferase enzymes, the specificity being determined by the prenylation motif in the protein substrate. These enzymes are protein:farnesyl transferase (FTase), which farnesylates proteins such as Ras and lamins; protein:geranylgeranyl transferase I (GGTase I), which geranylgeranylates mainly small GTPase proteins of the Rho family (e.g., Rho, Rac, and Cdc42); and Rab protein:geranylgeranyl transferase (Rab GGTase), which specifically geranylgeranylates small GTPases of the Rab family [2] that are involved in the trafficking of intracellular vesicles [3]. We recently identified 3-PEHPC [2-(3-pyridinyl)-1-hydroxyethylidene-1,1-phosphonocarboxylic acid; previously referred to as NE10790], a phosphonocarboxylate (PC) compound (Fig. 1), as the first specific inhibitor of Rab GGTase. 3-PEHPC prevents the prenylation of Rab proteins in vitro, without affecting prenylation of Ras or Rho family proteins [4]. However, the effect on the membrane association of Rab proteins following inhibition of Rab prenylation by 3-PEHPC or RIS has not been studied.
3-PEHPC is an analogue of the bisphosphonate drug risedronate (RIS), in which one of the phosphonate groups is replaced by a carboxylate group. Bisphosphonates (BPs), in particular those containing nitrogen (N-BPs), are the most widely used and effective treatments for diseases of bone metabolism characterized by excessive bone resorption, including post-menopausal osteoporosis, Paget's disease and tumor-associated osteolysis [5]. These drugs act by directly and selectively inhibiting the activity of osteoclasts, the cells responsible for bone resorption. Following in vivo administration, BPs rapidly target bone mineral due to their P-C-P backbone structure, which provides these drugs with a high affinity for calcium ions (reviewed by Rogers et al. [6]). During bone resorption, BPs are liberated from the bone surface and internalized by osteoclasts [7], [8]. N-BPs disrupt osteoclast function by inhibiting farnesyl diphosphate (FPP) synthase, blocking the synthesis of both farnesyl and geranylgeranyl isoprenoids and thereby preventing the prenylation of small GTPases required for osteoclast function [9], [10]. By inhibiting FPP synthase, N-BPs block prenylation of all small GTPases, although there is evidence that the effects of N-BPs on osteoclasts are mainly due to loss of geranylgeranylation of Rho family GTPases that are prenylated by GGTase I [11], [12], [13].
Like N-BPs, 3-PEHPC also inhibits bone resorption in vitro and in vivo, [4], [14], [15], indicating that, in addition to Rho proteins, prenylated Rabs are also crucial for osteoclast function [4]. However, 3-PEHPC is a much weaker inhibitor of bone resorption than RIS, probably due in part to the reduced affinity of 3-PEHPC for bone compared to RIS, as a result of the replacement of one of the phosphonate groups with a carboxylate group [15]. Currently, little is known about the relationship between molecular structure and inhibition of Rab GGTase by 3-PEHPC or other PCs [4]. By contrast, structure–activity studies using BPs in several different model systems has yielded valuable information regarding the structural features of BPs essential for their activity and potency for inhibiting FPP synthase. For example, as well as being important for binding to bone, the presence of two phosphonate groups is required for interaction with the molecular target [14], [15], [16], emphasized by the inability of 3-PEHPC to inhibit FPP synthase [4]. By contrast, a geminal hydroxyl group in nitrogen-containing BPs increases bone affinity [17], but does not appear to influence the ability of BPs to act at the cellular level [18]. Of most importance regarding potency, however, is the structure of the R2 side chain. Potent N-BPs are all characterized by the presence of a nitrogen in either an alkyl (e.g., alendronate) or heterocyclic (e.g., RIS) side chain. The 3-dimensional orientation of this basic nitrogen moiety is critical for effective inhibition of FPP synthase [10], since minor modifications to the side chain that alter the position of the nitrogen in relation to the P-C-P backbone can dramatically affect potency [10], [16], [19].
In this study, we examined the effect of RIS and 3-PEHPC on Rab prenylation in vitro and in vivo, and on the association of Rabs and other small GTPases with specific subcellular membranes. Since 3-PEHPC could represent a new class of pharmacological agents for inhibiting Rab prenylation, we also examined the role of molecular structure in inhibition of Rab GGTase and inhibition of bone resorption by PCs.
Section snippets
Reagents
RIS, 3-PEHPC, 3-PEPC [2-(3-pyridinyl)-1-ethylidene-1,1-phosphonocarboxylic acid], and 2-PEPC [2-(2-pyridinyl)-1-ethylidene-1,1-phosphonocarboxylic acid; Fig. 1] were from Procter & Gamble Pharmaceuticals, Cincinnati, OH (the synthesis of 3-PEPC and 2-PEPC will be described elsewhere). The drugs were dissolved in PBS, the pH adjusted to 7.4 with 1 N NaOH, then filter-sterilized by using a 0.2-μm filter. Mevastatin was purchased from Sigma Chemical Co. and converted from the lactone as described
3-PEHPC and RIS cause accumulation of unprenylated Rab proteins in J774 cells and osteoclasts
The ability to separate prenylated and unprenylated Rab proteins in cell lysates using Triton X-114 fractionation was first confirmed by metabolically labeling J774 cells with [14C]mevalonic acid lactone. As expected, radiolabeled, prenylated Rabs of molecular mass ∼25 kDa (Fig. 2A, top panel) and radiolabeled isoprenoid lipids that had not been incorporated into prenylated proteins (Fig. 2A, bottom panel) localized exclusively to the detergent-rich phase.
Western blot analysis of cell lysates
Discussion
We and others recently demonstrated that nitrogen-containing BPs (e.g., RIS), a blockbuster class of drugs used for the treatment of diseases characterized by excessive osteoclast-mediated bone resorption, such as osteoporosis [6], act by inhibiting FPP synthase [9], [10], [29]. Inhibition of FPP synthase by N-BPs prevents synthesis of the isoprenoid lipids, FPP, and GGPP, thereby inhibiting post-translational prenylation of proteins, including small GTPases of the Ras, Rho, and Rab families [4]
Acknowledgments
This work has been supported by project grants from the Scottish Hospital Endowments Research Trust and the Arthritis Research Campaign, and by Procter & Gamble Pharmaceuticals.
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