Thangaiah Subramanian

A Tagging-via-substrate Approach to Detect the Farnesylated Proteome Using Two-dimensional Electrophoresis Coupled with Western Blotting

Prenylation is a post-translational modification critical for the proper function of multiple physiologically important proteins, including small G-proteins, such as Ras. Methods allowing rapid and selective detection of protein farnesylation and geranylgeranylation are fundamental for the understanding of prenylated protein function and for monitoring efficacy of drugs such as farnesyltransferase inhibitors (FTIs). Although the natural substrates for prenyltransferases are farnesyl pyrophosphate and geranylgeranyl pyrophosphate, farnesyltransferase has been shown to incorporate isoprenoid analogues into protein substrates. In this study, protein prenyltransferase targets were labeled using anilinogeraniol, the alcohol precursor to the unnatural farnesyl pyrophosphate analogue 8-anilinogeranyl diphosphate in a tagging-via-substrate approach. Antibodies specific for the anilinogeranyl moiety were used to detect the anilinogeranyl-modified proteins. Coupling this highly effective labeling/detection method with two-dimensional electrophoresis and subsequent Western blotting allowed simple, rapid analysis of the complex farnesylated proteome. For example, this method elucidated the differential effects induced by two chemically distinct FTIs, BMS-214,662 and L-778,123. Although both FTIs strongly inhibited farnesylation of many proteins such as Lamins, NAP1L1, N-Ras, and H-Ras, only the dual prenylation inhibitor L-778,123 blocked prenylation of Pex19, RhoB, K-Ras, Cdc42, and Rap1. This snapshot approach has significant advantages over traditional techniques, including radiolabeling, anti-farnesyl antibodies, or mass spectroscopy, and enables dynamic analysis of the farnesylated proteome.

Protein Farnesyltransferase Catalyzed Isoprenoid Transfer To Peptide Depends On Lipid Size and Shape, not Hydrophobicity

Protein farnesyl transferase (FTase) catalyzes transfer of a 15‐carbon farnesyl group from farnesyl diphosphate (FPP) to a conserved cysteine in the C‐terminal Ca1a2X motif of a range of proteins, including the oncoprotein H‐Ras (“C” refers to the cysteine, “a” to any aliphatic amino acid, and “X” to any amino acid) and the lipid chain interacts with, and forms part of the Ca1a2X peptide binding site. Previous studies have shown that H‐Ras biological function is ablated when it is modified with lipids that are 3–5 orders of magnitude less hydrophobic than FPP. Here, we employed a library of anilinogeranyl diphosphate (AGPP) and phenoxygeranyl diphosphate (PGPP) derivatives with a range of polarities (log P (lipid alcohol)=0.7–6.8, log P (farnesol)=6.1) and shapes to examine whether FTase‐catalyzed transfer to peptide is dependent on the hydrophobicity of the lipid. Analysis of steady‐state transfer kinetics for analogues to dansyl–GCVLS peptide revealed that the efficiency of lipid transfer was highly dependent on both the shape and size, but was independent of the polarity of the analogue. These observations indicate that hydrophobic features of isoprenoids critical for their association with membranes and/or protein receptors are not required for efficient transfer to Ca1a2X peptides by FTase. Furthermore, the results of these studies indicate that the role played by the farnesyl lipid in the FTase mechanism is primarily structural. To explain these results we propose a model in which the FTase active site stabilizes a membrane interface‐like environment.

Functional Characterization of the Atypical Integral Membrane Lipid Phosphatase PDP1/PPAPDC2 Identifies a Pathway for Interconversion of Isoprenols and Isoprenoid Phosphates in Mammalian Cells

The polyisoprenoid diphosphates farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are intermediates in the synthesis of cholesterol and related sterols by the mevalonate pathway and precursors for the addition of isoprenyl anchors to many membrane proteins. We developed tandem mass spectrometry assays to evaluate polyisoprenoid diphosphate phosphatase activity of an unusual integral membrane lipid enzyme: type 1 polyisoprenoid diphosphate phosphatase encoded by the PPAPDC2 gene (PDP1/PPAPDC2). In vitro, recombinant PDP1/PPAPDC2 preferentially hydrolyzed polyisoprenoid diphosphates, including FPP and GGPP over a variety of glycerol- and sphingo-phospholipid substrates. Overexpression of mammalian PDP1/PPAPDC2 in budding yeast depletes cellular pools of FPP leading to growth defects and sterol auxotrophy. In mammalian cells, PDP1/PPAPDC2 localizes to the endoplasmic reticulum and nuclear envelope and, unlike the structurally related lipid phosphate phosphatases, is predicted to be oriented with key residues of its catalytic domain facing the cytoplasmic face of the membrane. Studies using synthetic isoprenols with chemical properties that facilitate detection by mass spectrometry identify a pathway for interconversion of isoprenols and isoprenoid diphosphates in intact mammalian cells and demonstrate a role for PDP1/PPAPDC2 in this process. Overexpression of PDP1/PPAPDC2 in mammalian cells substantially decreases protein isoprenylation and results in defects in cell growth and cytoskeletal organization that are associated with dysregulation of Rho family GTPases. Taken together, these results focus attention on integral membrane lipid phosphatases as regulators of isoprenoid phosphate metabolism and suggest that PDP1/PPAPDC2 is a functional isoprenoid diphosphate phosphatase.

Hydrophilic Anilinogeranyl Diphosphate Prenyl Analogues Are Ras Function Inhibitors

Sequential processing of H-Ras by protein farnesyl transferase (FTase), Ras converting enzyme (Rce1), and protein-S-isoprenylcysteine O-methyltransferase (Icmt) to give H-Ras C-terminal farnesyl-S-cysteine methyl ester is required for appropriate H-Ras membrane localization and function, including activation of the mitogen-activated protein kinase (MAPK) cascade. We employed a Xenopus laevis oocyte whole-cell model system to examine whether anilinogeranyl diphosphate analogues of similar shape and size, but with a hydrophobicity different from that of the FTase substrate farnesyl diphosphate (FPP), could ablate biological function of H-Ras. Analysis of oocyte maturation kinetics following microinjection of in vitro analogue-modified H-Ras into isoprenoid-depleted oocytes revealed that analogues with a hydrophobicity near that of FPP supported H-Ras biological function, while the analogues p-nitroanilinogeranyl diphosphate (p-NO2-AGPP), p-cyanoanilinogeranyl diphosphate (p-CN-AGPP), and isoxazolaminogeranyl diphosphate (Isox-GPP) with hydrophobicities 2-5 orders of magnitude lower than that of FPP did not. We found that although H-Ras modified with FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP was an efficient substrate for C-terminal postprenylation processing by Rce1 and Icmt, co-injection of H-Ras with analogues p-NO2-AGPP, p-CN-AGPP, or Isox-GPP could not activate MAPK. We propose that H-Ras biological function requires a minimum lipophilicity of the prenyl group to allow important interactions downstream of the C-terminal processed H-Ras protein. The hydrophilic FPP analogues p-NO2-AGPP, p-CN-AGPP, and Isox-GPP are H-Ras function inhibitors (RFIs) and serve as lead compounds for a unique class of potential anticancer therapeutics.

Efficient Use of Exogenous Isoprenols for Protein Isoprenylation by MDA-MB-231 Cells Is Regulated Independently of the Mevalonate Pathway

Background: Stable isotope/chemical probe mass spectrometry was used to monitor cancer cell metabolism of exogenous isoprenols. Results: Efficient use of exogenous isoprenols for protein isoprenylation was undiminished by genetic or pharmacological inhibition of HMG-CoA reductase. Conclusion: Exogenous isoprenols are metabolized independently of the mevalonate pathway. Significance: This study identifies and quantitates a pathway of isoprenol metabolism with potential relevance to cancer progression. Mammalian cells can use exogenous isoprenols to generate isoprenoid diphosphate substrates for protein isoprenylation, but the mechanism, efficiency, and biological importance of this process are not known. We developed mass spectrometry-based methods using chemical probes and newly synthesized stable isotope-labeled tracers to quantitate incorporation of exogenously provided farnesol, geranylgeraniol, and unnatural analogs of these isoprenols containing an aniline group into isoprenoid diphosphates and protein isoprenylcysteines by cultured human cancer cell lines. We found that at exogenous isoprenol concentrations >10 μm, this process can generate as much as 50% of the cellular isoprenoid diphosphate pool used for protein isoprenylation. Mutational activation of p53 in MDA-MB-231 breast cancer cells up-regulates the mevalonate pathway to promote tumor invasiveness. p53 silencing or pharmacological inhibition of HMG-CoA reductase in these cells decreases protein isoprenylation from endogenously synthesized isoprenoids but enhances the use of exogenous isoprenols for this purpose, indicating that this latter process is regulated independently of the mevalonate pathway. Our observations suggest unique opportunities for design of cancer cell-directed therapies and may provide insights into mechanisms underlying pleiotropic therapeutic benefits and unwanted side effects of mevalonate pathway inhibition.

Inhibitors of protein geranylgeranyltransferase-I lead to prelamin A accumulation in cells by inhibiting ZMPSTE24.

Protein farnesyltransferase (FTase) inhibitors, generally called “FTIs,” block the farnesylation of prelamin A, inhibiting the biogenesis of mature lamin A and leading to an accumulation of prelamin A within cells. A recent report found that a GGTI, an inhibitor of protein geranylgeranyltransferase-I (GGTase-I), caused an exaggerated accumulation of prelamin A in the presence of low amounts of an FTI. This finding was interpreted as indicating that prelamin A can be alternately prenylated by GGTase-I and that inhibiting both protein prenyltransferases leads to more prelamin A accumulation than blocking FTase alone. Here, we tested an alternative hypothesis—GGTIs are not specific for GGTase-I, and they lead to prelamin A accumulation by inhibiting ZMPSTE24 (a zinc metalloprotease that converts farnesyl–prelamin A to mature lamin A). In our studies, commonly used GGTIs caused prelamin A accumulation in human fibroblasts, but the prelamin A in GGTI-treated cells exhibited a more rapid electrophoretic mobility than prelamin A from FTI-treated cells. The latter finding suggested that the prelamin A in GGTI-treated cells might be farnesylated (which would be consistent with the notion that GGTIs inhibit ZMPSTE24). Indeed, metabolic labeling studies revealed that the prelamin A in GGTI-treated fibroblasts is farnesylated. Moreover, biochemical assays of ZMPSTE24 activity showed that ZMPSTE24 is potently inhibited by a GGTI. Our studies show that GGTIs inhibit ZMPSTE24, leading to an accumulation of farnesyl–prelamin A. Thus, caution is required when interpreting the effects of GGTIs on prelamin A processing.

Formation of a Novel Macrocyclic Alkaloid from the Unnatural Farnesyl Diphosphate Analogue Anilinogeranyl Diphosphate by 5-Epi-Aristolochene Synthase.

As part of an effort to identify substrate analogs suitable for helping to resolve structural features important for terpene synthases, the inhibition of 5-epi-aristolochene biosynthesis from farnesyl diphosphate (FPP) by the tobacco 5-epi-aristolochene synthase incubated with anilinogeranyl diphosphate (AGPP) was examined. The apparent noncompetitive nature of the inhibition supported further assessment of how AGPP might be bound to crystallographic forms of the enzyme. Surprisingly, the bound form of the inhibitor appeared to have undergone a cyclization event consistent with the native mechanism associated with FPP catalysis. Biocatalytic formation of a novel 13-membered macrocyclic paracyclophane alkaloid was confirmed by high-resolution GC-MS and NMR analysis. This work provides insights into new biosynthetic means for generating novel, functionally diversified, medium-sized terpene alkaloids.

Farnesyl diphosphate analogues with aryl moieties are efficient alternate substrates for protein farnesyltransferase.

Farnesylation is an important post-translational modification essential for the proper localization and function of many proteins. Transfer of the farnesyl group from farnesyl diphosphate (FPP) to proteins is catalyzed by protein farnesyltransferase (FTase). We employed a library of FPP analogues with a range of aryl groups substituting for individual isoprene moieties to examine some of the structural and electronic properties of the transfer of an analogue to the peptide catalyzed by FTase. Analysis of steady-state kinetics for modification of peptide substrates revealed that the multiple-turnover activity depends on the analogue structure. Analogues in which the first isoprene is replaced with a benzyl group and an analogue in which each isoprene is replaced with an aryl group are good substrates. In sharp contrast with the steady-state reaction, the single-turnover rate constant for dansyl-GCVLS alkylation was found to be the same for all analogues, despite the increased chemical reactivity of the benzyl analogues and the increased steric bulk of other analogues. However, the single-turnover rate constant for alkylation does depend on the Ca(1)a(2)X peptide sequence. These results suggest that the isoprenoid transition-state conformation is preferred over the inactive E·FPP·Ca(1)a(2)X ternary complex conformation. Furthermore, these data suggest that the farnesyl binding site in the exit groove may be significantly more selective for the farnesyl diphosphate substrate than the active site binding pocket and therefore might be a useful site for the design of novel inhibitors.

Structure and specificity of a permissive bacterial C-prenyltransferase.

This study highlights the biochemical and structural characterization of the L-tryptophan C-6 C-prenyltransferase PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including the antibiotic daptomycin (Cubicin®). This study also highlights two additional PTs (FgaPT2 and CdpNPT) as catalysts for daptomycin prenylation where novel prenylated daptomycins also displayed improved antibacterial activities over the parent drug.

Use of Synthetic Isoprenoids to Target Protein Prenylation and Rho GTPases in Breast Cancer Invasion

Dysregulation of Ras and Rho family small GTPases drives the invasion and metastasis of multiple cancers. For their biological functions, these GTPases require proper subcellular localization to cellular membranes, which is regulated by a series of post-translational modifications that result in either farnesylation or geranylgeranylation of the C-terminal CAAX motif. This concept provided the rationale for targeting farnesyltransferase (FTase) and geranylgeranyltransferases (GGTase) for cancer treatment. However, the resulting prenyl transferase inhibitors have not performed well in the clinic due to issues with alternative prenylation and toxicity. As an alternative, we have developed a unique class of potential anti-cancer therapeutics called Prenyl Function Inhibitors (PFIs), which are farnesol or geranyl-geraniol analogs that act as alternate substrates for FTase or GGTase. Here, we test the ability of our lead PFIs, anilinogeraniol (AGOH) and anilinofarnesol (AFOH), to block the invasion of breast cancer cells. We found that AGOH treatment effectively decreased invasion of MDA-MB-231 cells in a two-dimensional (2D) invasion assay at 100 µM while it blocked invasive growth in three-dimensional (3D) culture model at as little as 20 µM. Notably, the effect of AGOH on 3D invasive growth was phenocopied by electroporation of cells with C3 exotransferase. To determine if RhoA and RhoC were direct targets of AGOH, we performed Rho activity assays in MDA-MB-231 and MDA-MB-468 cells and found that AGOH blocked RhoA and RhoC activation in response to LPA and EGF stimulation. Notably, the geranylgeraniol analog AFOH was more potent than AGOH in inhibiting RhoA and RhoC activation and invasive growth. Interestingly, neither AGOH nor AFOH impacted 3D growth of MCF10A cells. Collectively, this study demonstrates that AGOH and AFOH dramatically inhibit breast cancer invasion, at least in part by blocking Rho function, thus, suggesting that targeting prenylation by using PFIs may offer a promising mechanism for treatment of invasive breast cancer.