A classic antibiotic reimagined: Rationally designed bacitracin variants exhibit potent activity against vancomycin-resistant pathogens.

Bacitracin is a macrocyclic peptide antibiotic that is widely used as a topical treatment for infections caused by gram-positive bacteria. Mechanistically, bacitracin targets bacteria by specifically binding to the phospholipid undecaprenyl pyrophosphate (C55PP), which plays a key role in the bacterial lipid II cycle. Recent crystallographic studies have shown that when bound to C55PP, bacitracin adopts a highly ordered amphipathic conformation. In doing so, all hydrophobic side chains align on one face of the bacitracin-C55PP complex, presumably interacting with the bacterial cell membrane. These insights led us to undertake structure-activity investigations into the individual contribution of the nonpolar amino acids found in bacitracin. To achieve this we designed, synthesized, and evaluated a series of bacitracin analogues, a number of which were found to exhibit significantly enhanced antibacterial activity against clinically relevant, drug-resistant pathogens. As for the natural product, these next-generation bacitracins were found to form stable complexes with C55PP. The structure-activity insights thus obtained serve to inform the design of C55PP-targeting antibiotics, a key and underexploited antibacterial strategy.

Mononuclear phagocyte morphological response to chemoattractants is dependent on geranylgeranyl pyrophosphate.

Statins are used to treat hypercholesterolemia and function by inhibiting the production of the rate-limiting metabolite mevalonate. As such, statin treatment not only inhibits de novo synthesis of cholesterol but also isoprenoids that are involved in prenylation, the posttranslational lipid modification of proteins. The immunomodulatory effects of statins are broad and often conflicting. Previous work demonstrated that statins increased survival and inhibited myeloid cell trafficking in a murine model of sepsis, but the exact mechanisms underlying this phenomenon were unclear. Herein, we investigated the role of prenylation in chemoattractant responses. We found that simvastatin treatment abolished chemoattractant responses induced by stimulation by C5a and FMLP. The inhibitory effect of simvastatin treatment was unaffected by the addition of either farnesyl pyrophosphate (FPP) or squalene but was reversed by restoring geranylgeranyl pyrophosphate (GGPP). Treatment with prenyltransferase inhibitors showed that the chemoattractant response to both chemoattractants was dependent on geranylgeranylation. Proteomic analysis of C15AlkOPP-prenylated proteins identified several geranylgeranylated proteins involved in chemoattractant responses, including RHOA, RAC1, CDC42, and GNG2. Chemoattractant responses in THP-1 human macrophages were also geranylgeranylation dependent. These studies provide data that help clarify paradoxical findings on the immunomodulatory effects of statins. Furthermore, they establish the role of geranylgeranylation in mediating the morphological response to chemoattractant C5a.NEW & NOTEWORTHY The immunomodulatory effect of prenylation is ill-defined. We investigated the role of prenylation on the chemoattractant response to C5a. Simvastatin treatment inhibits the cytoskeletal remodeling associated with a chemotactic response. We showed that the chemoattractant response to C5a was dependent on geranylgeranylation, and proteomic analysis identified several geranylgeranylated proteins that are involved in C5a receptor signaling and cytoskeletal remodeling. Furthermore, they establish the role of geranylgeranylation in mediating the response to chemoattractant C5a.

Farnesyl pyrophosphate is a new danger signal inducing acute cell death.

Cell death is a vital event in life. Infections and injuries cause lytic cell death, which gives rise to danger signals that can further induce cell death, inflammation, and tissue damage. The mevalonate (MVA) pathway is an essential, highly conserved and dynamic metabolic pathway. Here, we discover that farnesyl pyrophosphate (FPP), a metabolic intermediate of the MVA pathway, functions as a newly identified danger signal to trigger acute cell death leading to neuron loss in stroke. Harboring both a hydrophobic 15-carbon isoprenyl chain and a heavily charged pyrophosphate head, FPP leads to acute cell death independent of its downstream metabolic pathways. Mechanistically, extracellular calcium influx and the cation channel transient receptor potential melastatin 2 (TRPM2) exhibit essential roles in FPP-induced cell death. FPP activates TRPM2 opening for ion influx. Furthermore, in terms of a mouse model constructing by middle cerebral artery occlusion (MCAO), FPP accumulates in the brain, which indicates the function of the FPP and TRPM2 danger signal axis in ischemic injury. Overall, our data have revealed a novel function of the MVA pathway intermediate metabolite FPP as a danger signal via transient receptor potential cation channels.

The glucocorticoid-induced leucine zipper mediates statin-induced muscle damage.

Statins, the most prescribed class of drugs for the treatment of hypercholesterolemia, can cause muscle-related adverse effects. It has been shown that the glucocorticoid-induced leucine zipper (GILZ) plays a key role in the anti-myogenic action of dexamethasone. In the present study, we aimed to evaluate the role of GILZ in statin-induced myopathy. Statins induced GILZ expression in C2C12 cells, primary murine myoblasts/myotubes, primary human myoblasts, and in vivo in zebrafish embryos and human quadriceps femoris muscle. Gilz induction was mediated by FOXO3 activation and binding to the Gilz promoter, and could be reversed by the addition of geranylgeranyl, but not farnesyl, pyrophosphate. Atorvastatin decreased Akt phosphorylation and increased cleaved caspase-3 levels in myoblasts. This effect was reversed in myoblasts from GILZ knockout mice. Similarly, myofibers isolated from knockout animals were more resistant toward statin-induced cell death than their wild-type counterparts. Statins also impaired myoblast differentiation, and this effect was accompanied by GILZ induction. The in vivo relevance of our findings was supported by the observation that gilz overexpression in zebrafish embryos led to impaired embryonic muscle development. Taken together, our data point toward GILZ as an essential mediator of the molecular mechanisms leading to statin-induced muscle damage.

Statins induce skeletal muscle atrophy via GGPP depletion-dependent myostatin overexpression in skeletal muscle and brown adipose tissue.

Myopathy is the major adverse effect of statins. However, the underlying mechanism of statin-induced skeletal muscle atrophy, one of statin-induced myopathy, remains to be elucidated. Myostatin is a negative regulator of skeletal muscle mass and functions. Whether myostatin is involved in statin-induced skeletal muscle atrophy remains unknown. In this study, we uncovered that simvastatin administration increased serum myostatin levels in mice. Inhibition of myostatin with follistatin, an antagonist of myostatin, improved simvastatin-induced skeletal muscle atrophy. Simvastatin induced myostatin expression not only in skeletal muscle but also in brown adipose tissue (BAT). Mechanistically, simvastatin inhibited the phosphorylation of forkhead box protein O1 (FOXO1) in C2C12 myotubes, promoting the nuclear translocation of FOXO1 and thereby stimulating the transcription of myostatin. In differentiated brown adipocytes, simvastatin promoted myostatin expression mainly by inhibiting the expression of interferon regulatory factor 4 (IRF4). Moreover, the stimulative effect of simvastatin on myostatin expression was blunted by geranylgeranyl diphosphate (GGPP) supplementation in both myotubes and brown adipocytes, suggesting that GGPP depletion was attributed to simvastatin-induced myostatin expression. Besides, the capacities of statins on stimulating myostatin expression were positively correlated with the lipophilicity of statins. Our findings provide new insights into statin-induced skeletal muscle atrophy. Graphical headlights 1. Simvastatin induces skeletal muscle atrophy via increasing serum myostatin levels in mice; 2. Simvastatin promotes myostatin expression in both skeletal muscle and brown adipose tissue through inhibiting GGPP production; 3. The stimulating effect of statins on myostatin expression is positively correlated with the lipophilicity of statins.

Anti-tumor effects of mevalonate pathway inhibition in ovarian cancer.

BACKGROUND: Ovarian cancer remains the most fatal gynecological malignancy. Current therapeutic options are limited due to late diagnosis in the majority of the cases, metastatic spread to the peritoneal cavity and the onset of chemo-resistance. Thus, novel therapeutic approaches are required. Statins and amino-bisphosphonates are inhibitors of the mevalonate pathway, which is a fundamental pathway of cellular metabolism, essential for cholesterol production and posttranslational protein farnesylation and geranylgeranylation. While this pathway has emerged as a promising treatment target in several human malignancies, its potential as a therapeutic approach in ovarian cancer is still not fully understood. METHODS: Human ovarian cancer cell lines (IGROV-1, A2780, A2780cis) were treated with increasing concentrations (0.5-100 µM) of statins (simvastatin, atorvastatin, rosuvastatin) and zoledronic acid. Effects on cell vitality and apoptosis were assessed using Cell Titer Blue®, Caspase 3/7 Glo®, clonogenic assays as well as cleaved poly (ADP-ribose) polymerase (cPARP) detection. The inhibition of the mevalonate pathway was confirmed using Western Blot of unprenylated Ras and Rap1a proteins. Quantitative real-time PCR and ELISA were used to analyze modulations on several key regulators of ovarian cancer tumorigenesis. RESULTS: The treatment of IGROV-1 and A2780 cells with statins and zoledronic acid reduced vitality (by up to 80%; p < 0.001) and induced apoptosis by up to 8-folds (p < 0.001) in a dose-dependent fashion. Rescue experiments using farnesyl pyrophosphate or geranylgeranyl pyrophosphate evidenced that blocked geranylgeranylation is the major underlying mechanism of the pro-apoptotic effects. Gene expression of the tumor-promoting cytokines and mediators, such as transforming growth factor (TGF)-ß1, vascular endothelial growth factor (VEGF), interleukin (IL)-8, and IL-6 were significantly suppressed by statins and zoledronic acid by up to 90% (p < 0.001). For all readouts, simvastatin was most potent of all agents used. Cisplatin-resistant A2780cis cells showed a relative resistance to statins and zoledronic acid. However, similar to the effects in A2780 cells, simvastatin and zoledronic acid significantly induced caspase 3/7 activation (6-folds; p < 0.001). CONCLUSION: Our in vitro findings point to promising anti-tumor effects of statins and zoledronic acid in ovarian cancer and warrant additional validation in preclinical and clinical settings.

Human Diploid Fibroblast Cell Lines - a Model System for Studying Immunodulatory Properties of Modern Immunobiological Drugs and Viruses.

The immunomodulatory properties of immunobiological drugs Glutoxim and Phosprenyl we well as vesicular stomatitis virus and inactivated tick-borne encephalitis vaccine virus were studied using human diploid fibroblast cell line from the collection of M. P. Chumakov Federal Research Center for Research and Development of Immunobiological Products. All tested preparations exhibited immunomodulatory activity in human diploid fibroblast cell line. Glutoxim in doses of 0.1 and 0.25 µg/ml stimulated production of IL-6 and IL-10 during 24-48 h of culturing, but did not stimulate production of IL-1ß. Phosprenyl, on the contrary, increased production of IL-1ß and the levels of IL-6 and IL-10. Vesicular stomatitis virus stimulated the production of IL-1ß, IL-6, and IL-10, while inactivated tick-borne encephalitis vaccine virus stimulated the production of cytokines IL-8 and IL-18. Immunomodulatory activity of inactivated tick-borne encephalitis vaccine virus was first demonstrated in the in vitro system.

The novel high-frequency variant of TRPV3 p.A628T in East Asians showing faster sensitization in response to chemical agonists.

TRPV3, a member of the thermosensitive Ca2+-permeable TRPV channel subfamily expressed in skin and sensory nerves, is also activated by chemical agonists such as 2-aminoethyl diphenylborinate (2-APB). Repetitive stimuli induce sensitization of TRPV3 activation, characterized by the cumulative increase in current amplitude and linearization of current-voltage relation (I/V curve). Through genomic analysis of various populations, we found non-rare TRPV3 mutation (p.A628T) in East Asian people with an allele frequency of 0.249 while 0.007 in Caucasian. Slope conductance of unitary channel was not different between WT and p.A628T. Whole-cell patch clamp study of wildtype TRPV3 (WT) and p.A628T overexpressed in HEK293T cells showed similar sensitization by the repetitive increase in temperature from 23 to 37 °C, while slightly higher sensitization to 43 °C in p.A628T. In contrast, the repetitive application of 2-APB (10 µM) or carvacrol (100 µM) induced faster sensitization in p.A628T than WT. However, 1 µM farnesyl pyrophosphate, an intrinsic lipid metabolite agonist, induced similar level of slow activations in WT and p.A628T. In Fura-2 microspectrofluorimetry, the 2-APB pulses induced a faster increase of [Ca2+]c in p.A628T than WT. In terms of ionic selectivity of channels, WT and p.A628T showed similar Ca2+ permeability (PCa/PNa) calculated from the reversal potential of I/V curves. Taken together, p.A628T shows faster sensitization to chemical agonists that are reflected as higher [Ca2+]c signaling. Based on the intriguing pharmacological sensitivity, the physiological implications of p.A628T in the East Asian population require further investigation.

Isoprenoids increase bovine endometrial stromal cell tolerance to the cholesterol-dependent cytolysin from Trueperella pyogenes.

Preventing postpartum uterine disease depends on the ability of endometrial cells to tolerate the presence of the bacteria that invade the uterus after parturition. Postpartum uterine disease and endometrial pathology in cattle are most associated with the pathogen Trueperella pyogenes. Trueperella pyogenes secretes a cholesterol-dependent cytolysin, pyolysin, which causes cytolysis by forming pores in the plasma membrane of endometrial stromal cells. The aim of the present study was to identify cell-intrinsic pathways that increase bovine endometrial stromal cell tolerance to pyolysin. Pyolysin caused dose-dependent cytolysis of bovine endometrial stromal cells and leakage of lactate dehydrogenase into supernatants. Cell tolerance to pyolysin was increased by inhibitors that target the mevalonate and cholesterol synthesis pathway, but not the mitogen-activated protein kinase, cell cycle, or metabolic pathways. Cellular cholesterol was reduced and cell tolerance to pyolysin was increased by supplying the mevalonate-derived isoprenoid farnesyl pyrophosphate, or by inhibiting farnesyl-diphosphate farnesyltransferase 1 or geranylgeranyl diphosphate synthase 1 to increase the abundance of farnesyl pyrophosphate. Supplying the mevalonate-derived isoprenoid geranylgeranyl pyrophosphate also increased cell tolerance to pyolysin, but independent of changes in cellular cholesterol. However, geranylgeranyl pyrophosphate inhibits nuclear receptor subfamily 1 group H receptors (NR1H, also known as liver X receptors), and reducing the expression of the genes encoding NR1H3 or NR1H2 increased stromal cell tolerance to pyolysin. In conclusion, mevalonate-derived isoprenoids increased bovine endometrial stromal cell tolerance to pyolysin, which was associated with reducing cellular cholesterol and inhibiting NR1H receptors.

The role of atorvastatin in suppressing tumor growth of uterine fibroids.

BACKGROUND: Medical therapeutic options remain quite limited for uterine fibroids treatment. Statins, competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, have anti-tumoral effects on multiple cancer types, however, little is known about their effects on uterine fibroids. METHODS: Initially, we conducted a retrospective study of 120 patients with uterine fibroids and hyperlipidemia from the Second Affiliated Hospital of Wenzhou Medical University. Then, we evaluated the effect of atorvastatin on proliferation and apoptosis both in immortalized uterine fibroids cells and primary uterine fibroids cells. Furthermore, the molecular mechanism by which atorvastatin suppressed uterine fibroids cell growth was explored. RESULTS: Our results showed that atorvastatin use for 1 or 2 years significantly suppressed growth of uterine fibroids. Atorvastatin inhibited the proliferation of immortalized and primary uterine fibroids cells in a dose and time-dependent manner and stimulated apoptosis of uterine fibroids cells by inducing caspase-3 activation, up-regulating Bim and down-regulating Bcl-2. Additionally, atorvastatin treatment suppressed phosphorylation of ERK1/2 and JNK. Furthermore, GGPP, a downstream lipid isoprenoid intermediate, significantly rescued the effect of atorvastatin. CONCLUSIONS: These results suggest that atorvastatin exerts anti-tumoral effects on uterine fibroids through inhibition of cell proliferation and induction of apoptosis in HMG-CoA-dependent pathway. Our results provide the first clinical and preclinical data on the use of atorvastatin as a promising nonsurgical treatment option for uterine fibroids.

Inhibition of the RhoA/Rho-associated, coiled-coil-containing protein kinase-1 pathway is involved in the therapeutic effects of simvastatin on pulmonary arterial hypertension.

BACKGROUND: Recent research has shown that statins improve pulmonary arterial hypertension (PAH), but their mechanisms of action are not fully understood. This study aimed to investigate the role of RhoA/ROCK1 regulation in the therapeutic effects of simvastatin on PAH. METHODS: For in vivo experiments, rats (N = 40) were randomly assigned to four groups: control, simvastatin, monocrotaline (MCT), and MCT + simvastatin. The MCT group and MCT + simvastatin groups received proline dithiocarbamate (50 mg/kg, i.p.) on the first day of the study. The MCT + simvastatin group received simvastatin (2 mg/kg) daily for 4 weeks, after which pulmonary arterial pressure was measured by right heart catheterization. The protein and mRNA levels of Rho and ROCK1 were measured by immunohistochemistry, Western blot, and PCR. For in vitro experiments, human pulmonary endothelial cells were divided into seven groups: control, simvastatin, monocrotaline pyrrole (MCTP), MCTP + simvastatin, MCTP + simvastatin + mevalonate, MCTP + simvastatin + farnesyl pyrophosphate (FPP), and MCTP + simvastatin + FPP + geranylgeranyl pyrophosphate (GGPP). After 72 h exposed to the drugs, the protein and mRNA levels of RhoA and ROCK1 were measured by Western blot and PCR. RESULTS: The MCT group showed increased mean pulmonary arterial pressure, marked vascular remodeling, and increased protein and mRNA levels of RhoA and ROCK1 compared to the other groups (P < 0.05). In vitro, the MCTP group showed a marked proliferation of vascular endothelial cells, as well as increased protein and mRNA levels of RhoA and ROCK1 compared to the MCTP + simvastatin group. The MCTP + simvastatin + mevalonate group, MCTP + simvastatin+ FPP group, and MCTP + simvastatin + FPP + GGPP group showed increased mRNA levels of RhoA and ROCK1, as well as increased protein levels of RhoA, compared to the MCTP + simvastatin group. CONCLUSIONS: Simvastatin improved vascular remodeling and inhibited the development of PAH. The effects of simvastatin were mediated by inhibition of RhoA/ROCK1. Simvastatin decreased RhoA/ROCK1 overexpression by inhibition of mevalonate, FPP, and GGPP synthesis.

Polyprenyl Phosphates Induce a High Humoral and Cellular Response to Immunization with Recombinant Proteins of the Replicative Complex of the Hepatitis C Virus.

The search for new adjuvants remains the critical task for the creation of hepatitis C vaccines due to the weak immunogenicity of biotechnological products. When immunizing mice with the recombinant proteins NS3 and NS5B of the hepatitis C virus (HCV), the adjuvant activity of three immunomodulators was compared. Phosprenyl® on the basis of polyprenyl phosphate (PPP), chemically synthesized analogue of the bacterial cell wall glucosaminyl muramyl dipeptide (GMDP), and IFN-α recombinant protein were tested. GMDP increased the activity of IgG1 antibodies 4-6 times but did not stimulate the production of IFN-γ; IFN-α has not shown any adjuvant properties. The introduction of recombinant HCV proteins together with PPP in low doses increased the activity of IgG2a isotype antibodies 4-7 times and increased IFN-γ secretion 3 times. Thus, it was first shown that PPP polarizes the immune response to Th1-type and is a promising adjuvant for the development of a vaccine against hepatitis C.

Isoprenoids responsible for protein prenylation modulate the biological effects of statins on pancreatic cancer cells.

BACKGROUND: Statin treatment of hypercholesterolemia is accompanied also with depletion of the mevalonate intermediates, including farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) necessary for proper function of small GTPases. These include Ras proteins, prevalently mutated in pancreatic cancer. In our study, we evaluated the effect of three key intermediates of the mevalonate pathway on GFP-K-Ras protein localization and the gene expression profile in pancreatic cancer cells after exposure to individual statins. METHODS: These effects were tested on MiaPaCa-2 human pancreatic cancer cells carrying a K-Ras activating mutation (G12C) after exposure to individual statins (20 µM). The effect of statins (atorvastatin, lovastatin, simvastatin, fluvastatin, cerivastatin, rosuvastatin, and pitavastatin) and mevalonate intermediates on GFP-K-Ras protein translocation was analyzed using fluorescence microscopy. The changes in gene expression induced in MiaPaCa-2 cells treated with simvastatin, FPP, GGPP, and their combinations with simvastatin were examined by whole genome DNA microarray analysis. RESULTS: All tested statins efficiently inhibited K-Ras protein trafficking from cytoplasm to the cell membrane of the MiaPaCa-2 cells. The inhibitory effect of statins on GFP-K-Ras protein trafficking was partially prevented by addition of any of the mevalonate pathway's intermediates tested. Expressions of genes involved in metabolic and signaling pathways modulated by simvastatin treatment was normalized by the concurrent addition of FPP or GGPP. K-Ras protein trafficking within the pancreatic cancer cells is effectively inhibited by the majority of statins; the inhibition is eliminated by isoprenoid intermediates of the mevalonate pathway. CONCLUSIONS: Our data indicate that the anticancer effects of statins observed in numerous studies to a large extent are mediated through isoprenoid intermediates of the mevalonate pathway, as they influence expression of genes involved in multiple intracellular pathways.

Simvastatin Inhibits Cell Proliferation and Migration in Human Anaplastic Thyroid Cancer.

Malignant human anaplastic thyroid cancer (ATC) is pertinacious to conventional therapies. The present study investigated the anti-cancer activity of simvastatin and its underlying regulatory mechanism in cultured ATC cells. Simvastatin (0-20 µM) concentration-dependently reduced cell viability and relative colony formation. Depletions of mevalonate (MEV) and geranylgeranyl pyrophosphate (GGpp) by simvastatin induced G1 arrest and increased apoptotic cell populations at the sub-G1 phase. Adding MEV and GGpp prevented the simvastatin-inhibited cell proliferation. Immunoblotting analysis illustrated that simvastatin diminished the activation of RhoA and Rac1 protein, and this effect was prevented by pre-treatment with MEV and GGpp. Simvastatin increased the levels of p21cip and p27kip proteins and reduced the levels of hyperphosphorylated-Rb, E2F1 and CCND1 proteins. Adding GGpp abolished the simvastatin-increased levels of p27kip protein, and the GGpp-caused effect was abolished by Skp2 inhibition. Introduction of Cyr61 siRNA into ATC cells prevented the epidermal growth factor (EGF)-enhanced cell migration. The EGF-induced increases of Cyr61 protein expression and cell migration were prevented by simvastatin. Taken together, these results suggest that simvastatin induced ATC proliferation inhibition through the deactivation of RhoA/Rac1 protein and overexpression of p21cip and p27kip, and migration inhibition through the abrogation of Cyr61 protein expression.

[Antiviral activity of phosprenyl and gamapren against infection caused by influenza a (H5N1) virus in cell culture].

The antiviral activity of Phosprenyl and Gamapren in vitro against highly pathogenic strain of avian influenza H5N1 virus was studied. Inoculation of the virus to the susceptible cell culture led to development of the cytopathogenic effect. Preliminary introduction of Phosprenyl and Gamapren an hour prior to infecting the cells with virus 10.0 TCID50 dose completely inhibited the cytopathogenic activity of the virus. At higher doses of virus (100.0 TCID50) significant inhibition of the infectious activity of the virus was observed: 70% of infected cells survived under the action of Phosprenyl, and 90% under the action of Gamapren. With the introduction of the preparations simultaneously with the infection of cells with virus at a dose of 10.0 TCID50 virtually 100% of infected cells survived, while in control cultures death of 100% of the cells occurred. After infection with the virus at a dose of 100.0 TCID50 Phosprenyl and Gamapren caused 50% protection of the cells. The antiviral effect of the drugs Phosprenyl and Gamapren may be associated not only with their virulicidal, but with anti-viral activity as well.

Mechanism of Germacradien-4-ol Synthase-Controlled Water Capture.

The sesquiterpene synthase germacradiene-4-ol synthase (GdolS) from Streptomyces citricolor is one of only a few known high-fidelity terpene synthases that convert farnesyl diphosphate (FDP) into a single hydroxylated product. Crystals of unliganded GdolS-E248A diffracted to 1.50 Å and revealed a typical class 1 sesquiterpene synthase fold with the active site in an open conformation. The metal binding motifs were identified as D(80)DQFD and N(218)DVRSFAQE. Some bound water molecules were evident in the X-ray crystal structure, but none were obviously positioned to quench a putative final carbocation intermediate. Incubations in H2(18)O generated labeled product, confirming that the alcohol functionality arises from nucleophilic capture of the final carbocation by water originating from solution. Site-directed mutagenesis of amino acid residues from both within the metal binding motifs and without identified by sequence alignment with aristolochene synthase from Aspergillus terreus generated mostly functional germacradien-4-ol synthases. Only GdolS-N218Q generated radically different products (∼50% germacrene A), but no direct evidence of the mechanism of incorporation of water into the active site was obtained. Fluorinated FDP analogues 2F-FDP and 15,15,15-F3-FDP were potent noncompetitive inhibitors of GdolS. 12,13-DiF-FDP generated 12,13-(E)-ß-farnesene upon being incubated with GdolS, suggesting stepwise formation of the germacryl cation during the catalytic cycle. Incubation of GdolS with [1-(2)H2]FDP and (R)-[1-(2)H]FDP demonstrated that following germacryl cation formation a [1,3]-hydride shift generates the final carbocation prior to nucleophilic capture. The stereochemistry of this shift is not defined, and the deuteron in the final product was scrambled. Because no clear candidate residue for binding of a nucleophilic water molecule in the active site and no significant perturbation of product distribution from the replacement of active site residues were observed, the final carbocation may be captured by a water molecule from bulk solvent.

Skin Metabolite, Farnesyl Pyrophosphate, Regulates Epidermal Response to Inflammation, Oxidative Stress, and Migration.

Skin produces cholesterol and a wide array of sterols and non-sterol mevalonate metabolites, including isoprenoid derivative farnesyl pyrophosphate (FPP). To characterize FPP action in epidermis, we generated transcriptional profiles of primary human keratinocytes treated with zaragozic acid (ZGA), a squalene synthase inhibitor that blocks conversion of FPP to squalene resulting in endogenous accumulation of FPP. The elevated levels of intracellular FPP resulted in regulation of epidermal differentiation and adherens junction signaling, insulin growth factor (IGF) signaling, oxidative stress response and interferon (IFN) signaling. Immunosuppressive properties of FPP were evidenced by STAT-1 downregulation and prominent suppression of its nuclear translocation by IFNγ. Furthermore, FPP profoundly downregulated genes involved in epidermal differentiation of keratinocytes in vitro and in human skin ex vivo. Elevated levels of FPP resulted in induction of cytoprotective transcriptional factor Nrf2 and its target genes. We have previously shown that FPP functions as ligand for the glucocorticoid receptor (GR), one of the major regulator of epidermal homeostasis. Comparative microarray analyses show significant but not complete overlap between FPP and glucocorticoid regulated genes, suggesting that FPP may have wider transcriptional impact. This was further supported by co-transfection and chromatin immunoprecipitation experiments where we show that upon binding to GR, FPP recruits ß-catenin and, unlike glucocorticoids, recruits co-repressor GRIP1 to suppress keratin 6 gene. These findings have many clinical implications related to epidermal lipid metabolism, response to glucocorticoid therapy as well as pleiotropic effects of cholesterol lowering therapeutics, statins. J. Cell. Physiol. 231: 2452-2463, 2016. © 2016 Wiley Periodicals, Inc.

Geranylgeranyl pyrophosphate performs as an endogenous regulator of adipocyte function via suppressing the LXR pathway.

Isoprenoids such as geranylgeranyl pyrophosphate (GGPP) influence various biological processes. Here we show that GGPP inhibits adipocyte differentiation via the liver X receptors (LXRs) pathway. Intracellular GGPP levels and GGPP synthase (Ggps) mRNA expression increases during adipocyte differentiation. Ggps expression also increases in white adipose tissue of obese mice. GGPP addition reduces the expression of adipogenic marker genes such as adipocyte fatty acid binding protein, peroxisome proliferator-activated receptor γ, and insulin-stimulated glucose uptake. Similarly, over-expressing Ggps inhibits adipocyte differentiation. In contrast, Ggps knockdown promotes adipocyte differentiation. Inhibition of adipocyte differentiation by GGPP was partially reduced by LXR agonist T0901317. Furthermore, Ggps knockdown up-regulates LXR target genes during adipocyte differentiation. These results suggest that GGPP may act as an endogenous regulator of adipocyte differentiation and maturation through a mechanism partially dependent on the LXR pathway.

Statins inhibit blastocyst formation by preventing geranylgeranylation.

STUDY HYPOTHESIS: Statins, inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase of the mevalonate pathway and prescription drugs that treat hypercholesterolemia, compromise preimplantation mouse development via modulation of HIPPO signaling. STUDY FINDING: HMG-CoA reductase activity is required for trophectoderm specification, namely blastocyst cavity formation and Yes-associated protein (YAP) nuclear localization, through the production of isoprenoid geranylgeranyl pyrophosphate (GGPP) and the action of geranylgeranyl transferase. WHAT IS KNOWN ALREADY: Previous studies have shown that treatment of mouse embryos with mevastatin prevents blastocyst formation, but how HMG-CoA reductase is involved in preimplantation development is unknown. HIPPO signaling regulates specification of the trophectoderm lineage of the mouse blastocyst by controlling the nuclear localization of YAP. In human cell lines, the mevalonate pathway regulates YAP to mediate self-renewal and survival through geranylgeranylation of RHO proteins. These studies suggest that in preimplantation development, statins may act through HIPPO pathway to interfere with trophectoderm specification and thereby inhibit blastocyst formation. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Eight-cell stage (E2.5) mouse embryos were treated in hanging drop culture with chemical agents, namely statins (lovastatin, atorvastatin, cerivastatin and pravastatin), mevalonic acid (MVA), cholesterol, squalene, farnesyl pyrophosphate (FPP), geranylgeranyl pyrophosphate (GGPP), geranylgeranyltransferase inhibitor GGTI-298, RHO inhibitor I, and squalene synthase inhibitor YM-53601, up to the late blastocyst stage (E4.5). Efficiency of blastocyst formation was assessed based on gross morphology and the measurement of the cavity size using an image analysis software. Effects on cell lineages and HIPPO signaling were analyzed using immunohistochemistry with confocal microscopy based on the expression patterns of the lineage-specific markers and the nuclear accumulation of YAP. Effects on cell lineages were also examined by quantitative RT-PCR based on the transcript levels of the lineage-specific marker genes. Data were analyzed using one-way ANOVA and two-sample t-test. MAIN RESULTS AND THE ROLE OF CHANCE: All four statins examined inhibited blastocyst formation. The adverse impact of statins was rescued by supplementation of MVA (P < 0.01) or GGPP (P < 0.01) but not squalene nor cholesterol. Blastocyst formation was also prevented by GGTI-298 (P < 0.01). These results indicate that HMG-CoA reductase activity is required for blastocyst formation mainly through the production of GGPP but not cholesterol. Inhibition of RHO proteins, known targets of geranylgeranylation, impaired blastocyst formation, which was not reversed by GGPP supplementation. Nuclear localization of YAP was diminished by statin treatment but fully restored by supplementation of MVA (P < 0.01) or GGPP (P < 0.01). This suggests that HIPPO signaling is regulated by GGPP-dependent mechanisms, possibly geranylgeranylation of RHO, to enable trophectoderm formation. YM-53601 prevented blastocyst formation (P < 0.01), but its adverse impact was not rescued by supplementation of squalene or cholesterol, suggesting that squalene synthesis inhibition was not the cause of blastocyst defects. LIMITATIONS, REASONS FOR CAUTION: Analyses were conducted on embryos cultured ex vivo, but they enable the determination of specific concentrations that impair embryo development which can be compared with drug concentrations in the reproductive tract when testing in vivo impact of statins through animal experimentations. Also, analyses were conducted in only one species, the mouse. Epidemiological studies on the effects of various types of statins on the fertility of women are necessary. WIDER IMPLICATIONS OF THE FINDINGS: Our study reveals how the mevalonate pathway is required for blastocyst formation and intersects with HIPPO pathway to provide a mechanistic basis for the embryotoxic effect of statins. This bears relevance for women who are taking statins while trying to conceive, since statins have potential to prevent the conceptus from reaching the blastocyst stage and to cause early conceptus demise. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by grants from the George F. Straub Trust of the Hawaii Community Foundation (13ADVC-60315 to V.B.A.) and the National Institutes of Health, USA (P20GM103457 to V.B.A.). The authors have no conflict of interest to declare.

Simvastatin Reduces Cancerogenic Potential of Renal Cancer Cells via Geranylgeranyl Pyrophosphate and Mevalonate Pathway.

Simvastatin is a cholesterol-lowering drug, inhibiting 3-hydroxy-3-methylglutaryl-coenzyme CoA (HMG-CoA) reductase. Previous studies have indicated the anticancerous effects of simvastatin. Here, we evaluated the anticancerous potential of simvastatin in renal cell carcinoma (RCC) cell lines. RCC occurs with an incidence of 2-3% of all cancer entities with high chemoresistance rate. Therefore, the understanding of underlying mechanisms for RCC activity and the development of alternative therapies are essential. Human RCC cell lines Caki-1 and KTC-26 were treated with simvastatin (16 or 33 µM) for 48 or 72 h. The effects of the downstream substrates mevalonate (MA), farnesyl pyrophosphate (FPP), and geranylgeranyl pyrophosphate (GGPP) were evaluated using add-back experiments. Cell growth was assessed using MTT assay. Apoptosis and cell cycle were analyzed by flow cytometry. Apoptosis-involved proteins were evaluated by Western blot. Simvastatin caused dose- and time-dependent inhibition of RCC cell growth by cell cycle arrest and apoptosis induction. Substitution of MA or GGPP abolished these effects to a large extent. These findings suggest that the antiproliferative effects of simvastatin are not only mediated through cholesterol deprivation but also by prenylation-associated mechanisms, thereby providing new insights into tumor-suppressive ability of simvastatin and into novel additive treatment options in the management of RCC.