We have investigated the mechanism of action of Aquifex aeolicus IspH [E-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) reductase], together with its inhibition, using a combination of site-directed mutagenesis (K M ; V max ), EPR and 1 H, 2 H, 13 C, 31 P, and 57 Fe-electron-nuclear double resonance (ENDOR) spectroscopy. On addition of HMBPP to an (unreactive) E126A IspH mutant, a reaction intermediate forms that has a very similar EPR spectrum to those seen previously with the HMBPP "parent" molecules, ethylene and allyl alcohol, bound to a nitrogenase FeMo cofactor. The EPR spectrum is broadened on 57 Fe labeling and there is no evidence for the formation of allyl radicals. When combined with ENDOR spectroscopy, the results indicate formation of an organometallic species with HMBPP, a π∕σ "metallacycle" or η 2 -alkenyl complex. The complex is poised to interact with H þ from E126 (and H124) in reduced wt IspH, resulting in loss of water and formation of an η 1 -allyl complex. After reduction, this forms an η 3 -allyl π-complex (i.e. containing an allyl anion) that on protonation (at C2 or C4) results in product formation. We find that alkyne diphosphates (such as propargyl diphosphate) are potent IspH inhibitors and likewise form metallacycle complexes, as evidenced by 1 H, 2 H, and 13 C ENDOR, where hyperfine couplings of approximately 6 MHz for 13 C and 10 MHz for 1 H, are observed. Overall, the results are of broad general interest because they provide new insights into IspH catalysis and inhibition, involving organometallic species, and may be applicable to other Fe 4 S 4 -containing proteins, such as IspG.enzyme inhibition | iron-sulfur protein | isoprenoid biosynthesis | nonmevalonate pathway E nzymes that catalyze the formation of isoprenoids are of interest as drug targets. There are two main pathways involved in the early steps in isoprenoid biosynthesis: The mevalonate pathway found in animals and in pathogens such as Staphylococcus aureus, Trypanosoma cruzi, and Leishmania spp. (the causative agents of staph infections, Chagas' disease and the leishmaniases), and the nonmevalonate or Rohmer pathway found in most pathogenic bacteria, as well as in the malaria parasite, Plasmodium falciparum (1). Both pathways lead to formation of the C 5 -isoprenoids isopentenyl diphosphate (IPP, 1) and dimethylallyl diphosphate (DMAPP, 2). In the later stages of isoprenoid biosynthesis, these C 5 -compounds then form the farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) used in protein prenylation, sterol, and carotenoid biosynthesis. Understanding how the enzymes catalyzing these "downstream" events function has led to a better understanding of e.g. how FPP synthase (2) and GGPP synthase function, and can be inhibited (3); the discovery that bisphosphonates have potent antiparasitic activity (4); the clinical use of amiodarone (a squalene oxidase and oxidosqualene cyclase inhibitor) against Chagas' disease (5; 6) and leishmaniasis (7); anticancer agents that inhibit both FPPS and GGPPS (8); as wel...

We report the results of a series of chemical, EPR, ENDOR, and HYSCORE spectroscopic investigations of the mechanism of action (and inhibition) of GcpE, E-1-hydroxy-2-methyl-but-2-enyl-4-diphosphate (HMBPP) synthase, also known as IspG, an Fe 4 S 4 cluster-containing protein. We find that the epoxide of HMBPP when reduced by GcpE generates the same transient EPR species as observed on addition of the substrate, 2-C-methyl-D-erythritol-2, 4-cyclo-diphosphate. ENDOR and HYSCORE spectra of these transient species (using 2 H, 13 C and 17 O labeled samples) indicate formation of an Fe-C-H containing organometallic intermediate, most likely a ferraoxetane. This is then rapidly reduced to a ferracyclopropane in which the HMBPP product forms an η 2 -alkenyl π-(or π∕σ) complex with the 4th Fe in the Fe 4 S 4 cluster, and a similar "metallacycle" also forms between isopentenyl diphosphate (IPP) and GcpE. Based on this metallacycle concept, we show that an alkyne (propargyl) diphosphate is a good (K i ∼ 300 nM) GcpE inhibitor, and supported again by EPR and ENDOR results (a 13 C hyperfine coupling of ∼7 MHz), as well as literature precedent, we propose that the alkyne forms another π∕σ metallacycle, an η 2 -alkynyl, or ferracyclopropene. Overall, the results are of broad general interest because they provide new mechanistic insights into GcpE catalysis and inhibition, with organometallic bond formation playing, in both cases, a key role.4Fe-4S protein | GcpE (IspG) | metallacycle

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We report the results of an in vitro screening assay targeting the intraerythrocytic form of the malaria parasite Plasmodium falciparum using a library of 560 prenyl-synthase inhibitors. Based on "growth-rescue" and enzyme-inhibition experiments, geranylgeranyl diphosphate synthase (GGPPS) is shown to be a major target for the most potent leads, BPH-703 and BPH-811, lipophilic analogs of the bone-resorption drugs zoledronate and risedronate. We determined the crystal structures of these inhibitors bound to a Plasmodium GGPPS finding that their head groups bind to the ½Mg 2þ 3 cluster in the active site in a similar manner to that found with their more hydrophilic parents, whereas their hydrophobic tails occupy a long-hydrophobic tunnel spanning both molecules in the dimer. The results of isothermal-titration-calorimetric experiments show that both lipophilic bisphosphonates bind to GGPPS with, on average, a ΔG of −9 kcal mol −1 , only 0.5 kcal mol −1 worse than the parent bisphosphonates, consistent with the observation that conversion to the lipophilic species has only a minor effect on enzyme activity. However, only the lipophilic species are active in cells. We also tested both compounds in mice, finding major decreases in parasitemia and 100% survival. These results are of broad general interest because they indicate that it may be possible to overcome barriers to cell penetration of existing bisphosphonate drugs in this and other systems by simple covalent modification to form lipophilic analogs that retain their enzyme-inhibition activity and are also effective in vitro and in vivo.M alaria, caused by Plasmodium spp., causes approximately 1 million deaths each year (1), and there are ever-present problems due to drug resistance (2). There is, therefore, a need for new drugs and drug leads. In earlier work, we and others found that the bisphosphonate class of drugs (3) used to treat bonerelated diseases-osteoporosis, Paget disease, and hypercalcemia due to malignancy-also inhibited the growth of a range of parasitic protozoa, including Trypanosoma cruzi (4, 5), Trypanosoma brucei (4, 6), Leishmania spp. (4,7,8), Toxoplasma gondii (4, 9), Cryptosporidium parvum (10, 11), Entamoeba histolytica (4, 12, 13), and Plasmodium spp. (4, 13-15). In the case of Plasmodium spp., the most potent inhibitors were not, however, the nitrogencontaining bisphosphonates such as zoledronate or risedronate (Scheme 1) used to treat bone diseases, but more lipophilic n-alkyl bisphosphonates (13). Their target in Plasmodium falciparum was not determined. However, more recently, a Plasmodium vivax geranylgeranyl diphosphate synthase (PvGGPPS) has been cloned, expressed, purified, and crystallized, and its three-dimensional structure determined (16). The enzyme is inhibited by bisphosphonates (16), so it seemed possible that it might be a target for the inhibitors discovered earlier. To investigate this possibility, we recently determined the IC 50 values for 25 bisphosphonates against PvGGPPS and compared the results for enzyme inhi...

IspG is a 4Fe4S protein involved in isoprenoid biosynthesis. Most bacterial IspGs contain two domains: a TIM barrel (A) and a 4Fe4S domain (B), but in plants and malaria parasites, there is a large insert domain (A*) whose structure and function are unknown. We show that bacterial IspGs function in solution as (AB) 2 dimers and that mutations in either both A or both B domains block activity. Chimeras harboring an A-mutation in one chain and a B-mutation in the other have 50% of the activity seen in wild-type protein, because there is still one catalytically active AB domain. However, a plant IspG functions as an AA*B monomer. We propose, using computational modeling and electron microscopy, that the A* insert domain has a TIM barrel structure that interacts with the A domain. This structural arrangement enables the A and B domains to interact in a “cup and ball” manner during catalysis, just as in the bacterial systems. EPR/HYSCORE spectra of reaction intermediate, product, and inhibitor ligands bound to both two and three domain proteins are identical, indicating the same local electronic structure, and computational docking indicates these ligands bridge both A and B domains. Overall, the results are of broad general interest because they indicate the insert domain in three-domain IspGs is a second TIM barrel that plays a structural role and that the pattern of inhibition of both two and three domain proteins are the same, results that can be expected to be of use in drug design.

Background/Aims: Immunosuppression is one of the hallmarks of cancer; however, its molecular mechanism remains unknown. In the present study, we sought to investigate the expression and activation of yes-associated protein 1 (YAP-1) and its roles in T cells within hepatocellular carcinoma (HCC). Methods: The expression and activation of YAP-1 were accessed by real-time PCR, immunohistochemistry staining, western blot, and flow cytometry. The potential regulation effect of YAP-1 on Regulatory T cells (Tregs) differentiation was predicted using bioinformatics tools and verified by in vitro studies. Results: Significant overexpression and activation of YAP-1 was detected within peripheral blood mononuclear cells and showed positive linear correlation to Treg percentage; it may serve as a valuable indicator of a bad prognosis. Using in vitro studies, we found that overexpression and activation of YAP-1 can promote naïve T cell polarization stimulation to Tregs by increasing the expression of TGFBR2. The YAP-1/TEADs DNA binding site was spotted within the promoter region of TGFBR2 and related to its transcription activity. YAP-1 acted as a co-activator of TGFBR2 transcription by binding directly to the TGFBR2 promoter through TEADs. Conclusion: Overexpression and activation of YAP-1 in HCC T cells can induce immunosuppression by promoting Treg differentiation via transcriptional enhancement of TGFBR2.

Notch controls skeletogenesis, but its role in the remodeling of adult bone remains conflicting. In mature mice, the skeleton can become osteopenic or osteosclerotic depending on the time point at which Notch is activated or inactivated. Using adult EGFP reporter mice, we find that Notch expression is localized to osteocytes embedded within bone matrix. Conditional activation of Notch signaling in osteocytes triggers profound bone formation, mainly due to increased mineralization, which rescues both age-associated and ovariectomy-induced bone loss and promotes bone healing following osteotomy. In parallel, mice rendered haploinsufficient in γ-secretase presenilin-1 (Psen1), which inhibits downstream Notch activation, display almost-absent terminal osteoblast differentiation. Consistent with this finding, pharmacologic or genetic disruption of Notch or its ligand Jagged1 inhibits mineralization. We suggest that stimulation of Notch signaling in osteocytes initiates a profound, therapeutically relevant, anabolic response.

BackgroundThe loss of tumor suppressor gene (TSG) function is a critical step in the pathogenesis of human lung cancer. RBM5 (RNA-binding motif protein 5, also named H37/LUCA-15) gene from chromosome 3p21.3 demonstrated tumor suppressor activity. However, the role of RBM5 played in the occurrence and development of lung cancer is still not well understood.MethodPaired non-tumor and tumor tissues were obtained from 30 adenocarcinomas. The expression of RBM5 mRNA and protein was examined by RT-PCR and Western blot. A549 cell line was used to determine the apoptotic function of RBM5 in vitro. A549 cells were transiently transfected with pcDNA3.1-RBM5. AnnexinV analysis was performed by flow cytometry. Expression of Bcl-2, cleaved caspase-3, caspase-9 and PAPP proteins in A549 lung cancer cells and the A549 xenograft BALB/c nude mice model was determined by Western blot. Tumor suppressor activity of RBM5 was also examined in the A549 xenograft model treated with pcDNA3.1-RBM5 plasmid carried by attenuated Salmonella typhi Ty21a.ResultThe expression of RBM5 mRNA and protein was decreased significantly in adenocarcinoma tissues compared to that in the non-tumor tissues. In addition, as compared to the vector control, a significant growth inhibition of A549 lung cancer cells was observed when transfected with pcDNA3.1-RBM5 as determined by cell proliferation assay. We also found that overexpression of RBM5 induced both early and late apoptosis in A549 cells using AnnexinV/PI staining as determined by flow cytometry. Furthermore, the expression of Bcl-2 protein was decreased, whereas the expression of cleaved caspase-3, caspase-9 and PARP proteins was significantly increased in the RBM5 transfected cells; similarly, expression of decreased Bcl-2 and increased cleaved caspase-3 proteins was also examined in the A549 xenograft model. More importantly, we showed that accumulative and stable overexpression of RBM5 in the A549 xenograft BALB/c nude mice model significantly inhibited the tumor growth rate in vivo as compared to that in the control.ConclusionOur study demonstrates that RBM5 can inhibit the growth of lung cancer cells and induce apoptosis both in vitro and in vivo, which suggests that RBM5 might be used as a potential biomarker or target for lung cancer diagnosis and chemotherapy. Moreover, we propose a novel animal model set up in BALB/c nude mice treated with attenuated Salmonella as a vector carrying plasmids to determine RBM5 function in vivo.