Inhibition Of GTPase Activity Research Articles

Dynasore Alleviates LPS-Induced Acute Lung Injury by Inhibiting NLRP3 Inflammasome-Mediated Pyroptosis.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are clinically severe respiratory disorders without available pharmacological therapies. Dynasore is a cell-permeable molecule that inhibits GTPase activity and exerts protective effects in several disease models. However, whether dynasore can alleviate lipopolysaccharide (LPS)-induced ALI is unknown. This study investigated the effect of dynasore on macrophage activation and explored its potential mechanisms in LPS-induced ALI in vitro and in vivo. Bone marrow-derived macrophages (BMDMs) were activated classically with LPS or subjected to NLRP3 inflammasome activation with LPS+ATP. A mouse ALI model was established by the intratracheal instillation (i.t.) of LPS. The expression of PYD domains-containing protein 3 (NLRP3), caspase-1, and gasdermin D (GSDMD) protein was detected by Western blots. Inflammatory mediators were analyzed in the cell supernatant, in serum and bronchoalveolar lavage fluid (BALF) by enzyme-linked immunosorbent assays. Morphological changes in lung tissues were evaluated by hematoxylin and eosin staining. F4/80, Caspase-1 and GSDMD distribution in lung tissue was detected by immunofluorescence. Dynasore downregulated nuclear factor (NF)-κB signaling and reduced proinflammatory cytokine production in vitro and inhibited the production and release of interleukin (IL)-1β, NLRP3 inflammasome activation, and macrophage pyroptosis through the Drp1/ROS/NLRP3 axis. Dynasore significantly reduced lung injury scores and proinflammatory cytokine levels in both BALF and serum in vivo, including IL-1β and IL-6. Dynasore also downregulated the co-expression of F4/80, caspase-1 and GSDMD in lung tissue. Collectively, these findings demonstrated that dynasore could alleviate LPS-induced ALI by regulating macrophage pyroptosis, which might provide a new therapeutic strategy for ALI/ARDS.

Impaired mitochondrial dynamics and removal of the damaged mitochondria in diabetic retinopathy.

Mitochondrial dynamic plays a major role in their quality control, and the damaged mitochondrial components are removed by autophagy. In diabetic retinopathy, mitochondrial fusion enzyme, mitofusin 2 (Mfn2), is downregulated and mitochondrial dynamic is disturbed resulting in depolarized and dysfunctional mitochondria. Our aim was to investigate the mechanism of inhibition of Mfn2, and its role in the removal of the damaged mitochondria, in diabetic retinopathy. Using human retinal endothelial cells, effect of high glucose (20mM) on the GTPase activity of Mfn2 and its acetylation were determined. Role of Mfn2 in the removal of the damaged mitochondria was confirmed by regulating its acetylation, or by Mfn2 overexpression, on autophagosomes- autolysosomes formation and the mitophagy flux. High glucose inhibited GTPase activity and increased acetylation of Mfn2. Inhibition of acetylation, or Mfn2 overexpression, attenuated decrease in GTPase activity and mitochondrial fragmentation, and increased the removal of the damaged mitochondria. Similar phenomenon was observed in diabetic mice; overexpression of sirtuin 1 (a deacetylase) ameliorated diabetes-induced inhibition of retinal Mfn2 and facilitated the removal of the damaged mitochondria. Acetylation of Mfn2 has dual roles in mitochondrial homeostasis in diabetic retinopathy, it inhibits GTPase activity of Mfn2 and increases mitochondrial fragmentation, and also impairs removal of the damaged mitochondria. Thus, protecting Mfn2 activity should maintain mitochondrial homeostasis and inhibit the development/progression of diabetic retinopathy.

Identification of anti-Mycobacterium tuberculosis agents targeting the interaction of bacterial division proteins FtsZ and SepFe.

Tuberculosis (TB) is one of the deadly diseases caused by Mycobacterium tuberculosis (Mtb), which presents a significant public health challenge. Treatment of TB relies on the combination of several anti-TB drugs to create shorter and safer regimens. Therefore, new anti-TB agents working by different mechanisms are urgently needed. FtsZ, a tubulin-like protein with GTPase activity, forms a dynamic Z-ring in cell division. Most of FtsZ inhibitors are designed to inhibit GTPase activity. In Mtb, the function of Z-ring is modulated by SepF, a FtsZ binding protein. The FtsZ/SepF interaction is essential for FtsZ bundling and localization at the site of division. Here, we established a yeast two-hybrid based screening system to identify inhibitors of FtsZ/SepF interaction in M. tuberculosis. Using this system, we found compound T0349 showing strong anti-Mtb activity but with low toxicity to other bacteria strains and mice. Moreover, we have demonstrated that T0349 binds specifically to SepF to block FtsZ/SepF interaction by GST pull-down, fluorescence polarization (FP), surface plasmon resonance (SPR) and CRISPRi knockdown assays. Furthermore, T0349 can inhibit bacterial cell division by inducing filamentation and abnormal septum. Our data demonstrated that FtsZ/SepF interaction is a promising anti-TB drug target for identifying agents with novel mechanisms.

Design and synthesis of fascaplysin derivatives as inhibitors of FtsZ with potent antibacterial activity and mechanistic study

The increase in antibiotic resistance has made it particularly urgent to develop new antibiotics with novel antibacterial mechanisms. Inhibition of bacterial cell division by disrupting filamentous temperature-sensitive mutant Z (FtsZ) function is an effective and promising approach. A series of novel fascaplysin derivatives with tunable hydrophobicity were designed and synthesized here. The in vitro bioactivity assessment revealed that these compounds could inhibit the tested Gram-positive bacteria including methicillin-resistant S. aureus (MRSA) (MIC = 0.049–25 μg/mL), B. subtilis (MIC = 0.024–12.5 μg/mL) and S. pneumoniae (MIC = 0.049–50 μg/mL). Among them, compounds B3 (MIC = 0.098 μg/mL), B6 (MIC = 0.098 μg/mL), B8 (MIC = 0.049 μg/mL) and B16 (MIC = 0.098 μg/mL) showed the best bactericidal activities against MRSA and no significant tendency to trigger bacterial resistance as well as rapid bactericidal properties. The cell surface integrity of bacteria was significantly disrupted by hydrophobic tails of fascaplysin derivatives. Further studies revealed that these highly active amphiphilic compounds showed low hemolytic activity and cytotoxicity to mammalian cells. Preliminary mechanistic exploration suggests that B3, B6, B8 and B16 are potent FtsZ inhibitors to promote FtsZ polymerization and inhibit GTPase activity of FtsZ, leading to the death of bacterial cells by inhibiting bacterial division. Molecular docking simulations and structure-activity relationship (SAR) study reveal that appropriate increase in the hydrophobicity of fascaplysin derivatives and the addition of additional hydrogen bonds facilitated their binding to FtsZ proteins. These amphiphilic fascaplysin derivatives could serve as a novel class of FtsZ inhibitors, which not only gives new prospects for the application of compounds containing this skeleton but also provides new ideas for the discovery of new antibiotics.

Direct Targeting KRAS Mutation in Non-Small Cell Lung Cancer: Focus on Resistance.

KRAS is the most frequently mutated oncogene in non-small cell lung cancers (NSCLC), with a frequency of around 30%, and encoding a GTPAse that cycles between active form (GTP-bound) to inactive form (GDP-bound). The KRAS mutations favor the active form with inhibition of GTPAse activity. KRAS mutations are often with poor response of EGFR targeted therapies. KRAS mutations are good predictive factor for immunotherapy. The lack of success with direct targeting of KRAS proteins, downstream inhibition of KRAS effector pathways, and other strategies contributed to a focus on developing mutation-specific KRAS inhibitors. KRAS p.G12C mutation is one of the most frequent KRAS mutation in NSCLC, especially in current and former smokers (over 40%), which occurs among approximately 12-14% of NSCLC tumors. The mutated cysteine resides next to a pocket (P2) of the switch II region, and P2 is present only in the inactive GDP-bound KRAS. Small molecules such as sotorasib are now the first targeted drugs for KRAS G12C mutation, preventing conversion of the mutant protein to GTP-bound active state. Little is known about primary or acquired resistance. Acquired resistance does occur and may be due to genetic alterations in the nucleotide exchange function or adaptative mechanisms in either downstream pathways or in newly expressed KRAS G12C mutation.

Identification of Potential Modulators of the RGS7/Gβ5/R7BP Complex.

Regulators of G protein signaling (RGS) proteins serve as critical regulatory nodes to limit the lifetime and extent of signaling via G protein-coupled receptors (GPCRs). Previously, approaches to pharmacologically inhibit RGS activity have mostly focused on the inhibition of GTPase activity by interrupting the interaction of RGS proteins with the G proteins they regulate. However, several RGS proteins are also regulated by association with binding partners. A notable example is the mammalian RGS7 protein, which has prominent roles in metabolic control, vision, reward, and actions of opioid analgesics. In vivo, RGS7 exists in complex with the binding partners type 5 G protein β subunit (Gβ5) and R7 binding protein (R7BP), which control its stability and activity, respectively. Targeting the whole RGS7/Gβ5/R7BP protein complex affords the opportunity to allosterically tune opioid receptor signaling following opioid engagement while potentially bypassing undesirable side effects. Hence, we implemented a novel strategy to pharmacologically target the interaction between RGS7/Gβ5 and R7BP. To do so, we searched for protein complex inhibitors using a time-resolved fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay that measures compound-mediated alterations in the FRET signal between RGS7/Gβ5 and R7BP. We performed two HTS campaigns, each screening ~100,000 compounds from the Scripps Drug Discovery Library (SDDL). Each screen yielded more than 100 inhibitors, which will be described herein.

Indole-core-based novel antibacterial agent targeting FtsZ.

BackgroundThe prevalence of drug-resistant bacterial infections urges the development of new antibacterial agents that possess a mechanism of action different from traditional antibiotics. FtsZ has been recognized as a key functional protein in bacterial cell division and it is currently believed to be a potential target for the development of novel antibacterial agents.PurposeThe primary aim of the study is to screen out an inhibitor targeting at FtsZ and followed to investigate its antibacterial activity and mode of action.MethodsCell-based cell division inhibitory screening assay, antimicrobial susceptibility test, minimum bactericidal concentration assay, time-killing curve determination, FtsZ polymerization assay, GTPase activity assay, and molecular modeling were performed in the present study.ResultsThe screening study from a small library consisting of benzimidazole and indole derivatives discovered a compound (CZ74) with an indole-core structure. The compound exhibited strong cell division inhibitory effect. In addition, CZ74 shows high antibacterial potency against a number of tested Gram-positive bacteria, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus. The minimum inhibitory concentration values obtained were within the range of 2–4 µg/mL. The results of biological study revealed that CZ74 at 2 µg/mL is able to disrupt FtsZ polymerization and inhibit GTPase activity and cell division. From molecular modeling study, CZ74 is found possibly binding into the interdomain cleft of FtsZ protein and then leads to inhibitory effects.ConclusionThis indole-cored molecule CZ74 could be a potential lead compound and could be further developed as a new generation of antibacterial agents targeting FtsZ to combat against multidrug-resistant bacteria.

Design, synthesis and antibacterial evaluation of 2,4-disubstituted-6-thiophenyl-pyrimidines

The increasing incidences of multidrug resistant bacterial infections urge the development of novel antibacterial having a new mechanism of action. The small molecule-based inhibitors targeting at the cell division protein FtsZ has been recognized as a promising approach to search for new antibacterial with high potency. In the present study, a series of novel 2,4-disubstituted-6-thiophenyl-pyrimidine derivatives were synthesized and their antibacterial activities against clinically related pathogens were investigated. The compounds show strong antibacterial activities against MRSA and VREs. The antibacterial activity of compound Bb2 against MRSA and VREs (MIC values: 2 μg/mL) is stronger than that of methicillin and vancomycin. From the in vitro and in vivo results, Bb2 was found to inhibit GTPase activity and FtsZ polymerization. The compound is able to inhibit bacterial cell division through interacting with GTP binding site of FtsZ and thus causing cell death. In addition, S. aureus was found to develop resistance to methicillin but not for Bb2, which was proved in our resistance generation experiments.

The peroxyl radical-induced oxidation of Escherichia coli FtsZ and its single tryptophan mutant (Y222W) modifies specific side-chains, generates protein cross-links and affects biological function

FtsZ (filamenting temperature-sensitive mutant Z) is a key protein in bacteria cell division. The wild-type Escherichia coli FtsZ sequence (FtsZwt) contains three tyrosine (Tyr, Y) and sixteen methionine (Met, M) residues. The Tyr at position 222 is a key residue for FtsZ polymerization. Mutation of this residue to tryptophan (Trp, W; mutant Y222W) inhibits GTPase activity resulting in an extended time in the polymerized state compared to FtsZwt. Protein oxidation has been highlighted as a determinant process for bacteria resistance and consequently oxidation of FtsZwt and the Y222W mutant, by peroxyl radicals (ROO•) generated from AAPH (2,2′-azobis(2-methylpropionamidine) dihydrochloride) was studied. The non-oxidized proteins showed differences in their polymerization behavior, with this favored by the presence of Trp at position 222. AAPH-treatment of the proteins inhibited polymerization. Protein integrity studies using SDS-PAGE revealed the presence of both monomers and oligomers (dimers, trimers and high mass material) on oxidation. Western blotting indicated the presence of significant levels of protein carbonyls. Amino acid analysis showed that Tyr, Trp (in the Y222W mutant), and Met were consumed by ROO•. Quantification of the number of moles of amino acid consumed per mole of ROO• shows that most of the initial oxidant can be accounted for at low radical fluxes, with Met being a major target. Western blotting provided evidence for di-tyrosine cross-links in the dimeric and trimeric proteins, confirming that oxidation of Tyr residues, at positions 339 and/or 371, are critical to ROO•-mediated crosslinking of both the FtsZwt and Y222W mutant protein. These findings are in agreement with di-tyrosine, N-formyl kynurenine, and kynurenine quantification assessed by UPLC, and with LC-MS data obtained for AAPH-treated protein samples.

Actions of quercetin, a flavonoid, on ion transporters: its physiological roles.

Flavonoids keep us healthy by controlling various body and cellular functions. It is well known that cations, such as Na+ , K+ , and Ca2+ , play important roles in the regulation of body and cellular functions, including generation of action potentials and the resting membrane potential of neural and muscle cells and signal transduction as intracellular second messengers. However, we have little information on the physiological roles of anions, particularly Cl- , in body and cellular functions. Quercetin, a flavonoid, stimulates Na+ -K+ -2Cl- cotransporter 1 (NKCC1), which is one of the most important ion transporters regulating the cytosolic Cl- concentration ([Cl- ]c ). Here, we introduce the molecular mechanism by which flavonoids, specifically quercetin, act through elevation of [Cl- ]c via activation of NKCC1 on important factors controlling various body and cellular functions, such as (1) antihypertensive actions controlling blood volume dependent on the amounts of renal Na+ reabsorption via expression of the epithelial Na+ channel, (2) neurite-elongating actions via polymerization of tubulin by inhibiting GTPase activity, and (3) antibacterial and antiviral infective actions through stimulation of epithelial Cl- secretion by increasing the driving force for epithelial Cl- secretion.

Effects of rhodomyrtone on Gram-positive bacterial tubulin homologue FtsZ.

Rhodomyrtone, a natural antimicrobial compound, displays potent activity against many Gram-positive pathogenic bacteria, comparable to last-defence antibiotics including vancomycin and daptomycin. Our previous studies pointed towards effects of rhodomyrtone on the bacterial membrane and cell wall. In addition, a recent molecular docking study suggested that the compound could competitively bind to the main bacterial cell division protein FtsZ. In this study, we applied a computational approach (in silico), in vitro, and in vivo experiments to investigate molecular interactions of rhodomyrtone with FtsZ. Using molecular simulation, FtsZ conformational changes were observed in both (S)- and (R)-rhodomyrtone binding states, compared with the three natural states of FtsZ (ligand-free, GDP-, and GTP-binding states). Calculations of free binding energy showed a higher affinity of FtsZ to (S)-rhodomyrtone (−35.92 ± 0.36 kcal mol−1) than the GDP substrate (−23.47 ± 0.25 kcal mol−1) while less affinity was observed in the case of (R)-rhodomyrtone (−18.11 ± 0.11 kcal mol−1). In vitro experiments further revealed that rhodomyrtone reduced FtsZ polymerization by 36% and inhibited GTPase activity by up to 45%. However, the compound had no effect on FtsZ localization in Bacillus subtilis at inhibitory concentrations and cells also did not elongate after treatment. Higher concentrations of rhodomyrtone did affect localization of FtsZ and also affected localization of its membrane anchor proteins FtsA and SepF, showing that the compound did not specifically inhibit FtsZ but rather impaired multiple divisome proteins. Furthermore, a number of cells adopted a bean-like shape suggesting that rhodomyrtone possibly possesses further targets involved in cell envelope synthesis and/or maintenance.

Dynasore Improves Motor Function Recovery via Inhibition of Neuronal Apoptosis and Astrocytic Proliferation after Spinal Cord Injury in Rats.

Spinal cord injury (SCI) is a common and devastating central nervous system insult which lacks efficient treatment. Our previous experimental findings indicated that dynamin-related protein 1 (Drp1) mediates mitochondrial fission during SCI, and inhibition of Drp1 plays a significant protective effect after SCI in rats. Dynasore inhibits GTPase activity at both the plasma membrane (dynamin 1, 2) and the mitochondria membrane (Drp1). The aim of the present study was to investigate the beneficial effects of dynasore on SCI and its underlying mechanism in a rat model. Sprague-Dawley rats were randomly assigned to sham, SCI, and 1, 10, and 30mg dynasore groups. The rat model of SCI was established using an established Allen's model. Dynasore was administered via intraperitoneal injection immediately. Results of motor functional test indicated that dynasore ameliorated the motor dysfunction greatly at 3, 7, and 10days after SCI in rats (P<0.05). Results of western blot showed that dynasore has remarkably reduced the expressions of Drp1, dynamin 1, and dynamin 2 and, moreover, decreased the Bax, cytochrome C, and active Caspase-3 expressions, but increased the expressions of Bcl-2 at 3days after SCI (P<0.05). Notably, the upregulation of proliferating cell nuclear antigen (PCNA) and glial fibrillary acidic protein (GAFP) are inhibited by dynasore at 3days after SCI (P<0.05). Results of immunofluorescent double labeling showed that there were less apoptotic neurons and proliferative astrocytes in the dynasore groups compared with SCI group (P<0.05). Finally, histological assessment via Nissl staining demonstrated that the dynasore groups exhibited a significantly greater number of surviving neurons compared with the SCI group (P<0.05). This neuroprotective effect was dose-dependent (P<0.05). To our knowledge, this is the first study to indicate that dynasore significantly enhances motor function which may be by inhibiting the activation of neuronal mitochondrial apoptotic pathway and astrocytic proliferation in rats after SCI.

Guanylate-Binding Protein 1, an Interferon-Induced GTPase, Exerts an Antiviral Activity against Classical Swine Fever Virus Depending on Its GTPase Activity

ABSTRACTMany viruses trigger the type I interferon (IFN) pathway upon infection, resulting in the transcription of hundreds of interferon-stimulated genes (ISGs), which define the antiviral state of the host. Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), a highly contagious viral disease endangering the pig industry in many countries. However, anti-CSFV ISGs are poorly documented. Here we screened 20 ISGs that are commonly induced by type I IFNs against CSFV in lentivirus-delivered cell lines, resulting in the identification of guanylate-binding protein 1 (GBP1) as a potent anti-CSFV ISG. We observed that overexpression of GBP1, an IFN-induced GTPase, remarkably suppressed CSFV replication, whereas knockdown of endogenous GBP1 expression by small interfering RNAs significantly promoted CSFV growth. Furthermore, we demonstrated that GBP1 acted mainly on the early phase of CSFV replication and inhibited the translation efficiency of the internal ribosome entry site of CSFV. In addition, we found that GBP1 was upregulated at the transcriptional level in CSFV-infected PK-15 cells and in various organs of CSFV-infected pigs. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown assays revealed that GBP1 interacted with the NS5A protein of CSFV, and this interaction was mapped in the N-terminal globular GTPase domain of GBP1. Interestingly, the K51 of GBP1, which is crucial for its GTPase activity, was essential for the inhibition of CSFV replication. We showed further that the NS5A-GBP1 interaction inhibited GTPase activity, which was critical for its antiviral effect. Taking our findings together, GBP1 is an anti-CSFV ISG whose action depends on its GTPase activity.IMPORTANCE Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an economically important viral disease affecting the pig industry in many countries. To date, only a few host restriction factors against CSFV, including interferon-stimulated genes (ISGs), have been characterized. Using a minilibrary of porcine ISGs, we identify porcine guanylate-binding protein 1 (GBP1) as a potent antiviral ISG against CSFV. We further show that the anti-CSFV action of GBP1 depends on its GTPase activity. The K51 of GBP1, critical for its GTPase activity, is essential for the antiviral action of GBP1 against CSFV replication, and the binding of the NS5A protein to GBP1 antagonizes the GTPase activity and thus the antiviral effect. This study will facilitate the development of anti-CSFV therapeutic agents by targeting host factors and may provide a new strategy for the control of CSF.

Abstract POSTER-BIOL-1320: Rho-family GTPases as therapeutic targets in ovarian cancer

Abstract Purpose of the study: Although Rac1 and Cdc42 are considered attractive therapeutic targets, no selective inhibitors of these GTPases are in clinical trials. The Ras-homologous (Rho) family GTPases Rac1 and Cdc42 contribute to metastatic dissemination through regulation of actin reorganization, cell motility, cell-cell and cell-extracellular matrix adhesion. Using high throughput screening and cheminformatics, we identified the R-enantiomer of ketorolac as a novel inhibitor of Rac1 and Cdc42. R-enantiomers of nonsteroidal anti-inflammatory drugs are poor inhibitors of cyclooxygenase (COX) activity, yet little is known about the pharmacologic activities or targets of the R-enantiomers. The purpose of this study was to investigate the effects of R-ketorolac on ovarian cancer. Experimental procedures: GTPase target expression and activity was determined by immunohistochemistry, RT-PCR and enzymatic assays. The effects of racemic, R- and S-ketorolac on proliferation, adhesion and migration were investigated using human ovarian tumor cells (OvCA 429 and SKOV3ip). In vivo effect of ketorolac treatments was determined in a xenograft model using SKOV3ip cells. Pharmacokinetic and pharmacodynamic assessments of racemic R/S-ketorolac (Toradol®) in patients were conducted in women with suspected advanced stage ovarian, fallopian tube or primary peritoneal cancer with planned optimal cytoreductive surgery. Ascites samples were obtained for measurement of cell adhesion and drug inhibition of GTPase activity. After placement of an IP port the recommended dose of IV racemic ketorolac was administered and blood and peritoneal fluid were obtained at T=0, 1h, 6h and 24h. R- and S-ketorolac concentrations in serum and peritoneal fluid were measured by HPLC. GTPase inhibitory activity of ketorolac was assessed in peritoneal tumor cells. Summary of the data: Elevation of Cdc42 protein and expression of the constitutively active Rac1b splice variant of Rac1 were detected in ovarian cancer specimens providing the first evidence for dysregulation of these GTPase targets in ovarian cancer. R-ketorolac, and not S-ketorolac, inhibits Rac1 and Cdc42 activity demonstrating an unexpected pharmacologic activity for the R-enantiomer. R-ketorolac, but not S-ketorolac, inhibits cell adhesion and migration, and reduced peritoneal tumor implantation in a mouse xenograft model. In the clinical studies using R/S-ketorolac for post-operative pain management, we found that ketorolac distributed to peritoneal fluids within 6 hours and fluids were highly enriched in the R-enantiomer compared to the S-enantiomer. Rac1 and Cdc42 activity was inhibited in ovarian tumor cells retrieved from the peritoneal cavity post-ketorolac administration. Cell adhesion was decreased by R-ketorolac in patient-derived ovarian tumor cells. Conclusions: The findings show R-ketorolac is a novel inhibitor of Rac1 and/or Cdc42, and active in ovarian cancer model systems. The favorable distribution of R-ketorolac in the peritoneal cavity coupled with GTPase inhibition in cells retrieved from the intraperitoneal compartment support the potential benefit of R-ketorolac for ovarian cancer patients. Citation Format: Hudson LG, Kenney SR, Guo Y, Adams S, Rutledge T, Muller CY, Wandinger-Ness A. Rho-family GTPases as therapeutic targets in ovarian cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1320.

Identification of Dynamin-2-Mediated Endocytosis as a New Target of Osteoporosis Drugs, Bisphosphonates

Nitrogen-containing bisphosphonates are pyrophosphate analogs that have long been the preferred prescription for treating osteoporosis. Although these drugs are considered inhibitors of prenylation and are believed to exert their effects on bone resorption by disrupting the signaling pathways downstream of prenylated small GTPases, this explanation seems to be insufficient. Because other classes of prenylation inhibitors have recently emerged as potential antiviral therapeutic agents, we first investigated here the effects of bisphosphonates on simian virus 40 and adenovirus infections and, to our surprise, found that viral infections are suppressed by bisphosphonates through a prenylation-independent pathway. By in-house affinity-capture techniques, dynamin-2 was identified as a new molecular target of bisphosphonates. We present evidence that certain bisphosphonates block endocytosis of adenovirus and a model substrate by inhibiting GTPase activity of dynamin-2. Hence, this study has uncovered a previously unknown mechanism of action of bisphosphonates and offers potential novel use for these drugs.

Coeliac autoantibodies can enhance transamidating and inhibit GTPase activity of tissue transglutaminase: Dependence on reaction environment and enzyme fitness

Modification of the enzymatic functions of tissue transglutaminase (TG2) by anti-TG2 autoantibodies may play a role in manifestations of coeliac disease. Our aim was to evaluate the effect of coeliac autoantibodies on reactions catalysed by TG2 by a systematic biochemical approach, and in relation to observed clinical presentation type. Coeliac antibodies did not have significant inhibitory effect on transamidation/deamidation activity of TG2 as measured by amine-incorporation into solid and immobilised casein and by ultraviolet kinetic assay. In contrast, immunoglobulins from patients with severe malabsorption enhanced the reaction velocity to 105.4–242.2%. This activating effect was dose-dependent, most pronounced with immobilised glutamine-acceptor substrates, and correlated inversely with the basal specific activity of the enzyme and with dietary treatment. A similar activation could be demonstrated also with the TG2-specific fraction of autoantibodies and in transamidation activity assays which use fibronectin-bound TG2 and thereby mimic in vivo conditions. These results suggest that coeliac antibodies may stabilise the enzyme in a catalytically advantageous conformation. GTPase activity of TG2 decreased to 67.0–73.4% in the presence of antibodies raising the possibility that inhibition of GTPase activity may affect cellular signalling in case coeliac autoantibodies would reach intracellular compartments.

New Insights into the Role of Conserved, Essential Residues in the GTP Binding/GTP Hydrolytic Cycle of Large G Proteins

The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the alpha subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of Galpha subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a Galpha chimera (designated alpha(T)*), comprised mainly of the alpha subunit of retinal transducin (alpha(T)) and the Switch III region from the alpha subunit of G(i1). As expected, both the alpha(T)*(R174C) and alpha(T)*(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and Gbetagamma. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and Gbetagamma to undergo GDP-[(35)S]GTPgammaS exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotide-bound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P(i) complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal Galpha subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.

GTPase Implicated in Bacterial DNA Damage Checkpoint

In eukaryotic cells, defects in replication initiate a checkpoint signaling pathway that halts cell division so that cells are protected from deadly effects of DNA damage. Although it makes sense that prokaryotes would also benefit from such a protective response, an equivalent signaling mechanism has not been defined. A possible component of such a pathway is described by Foti et al. , who used a genetic screen in Escherichia coli to identify mutations that increased sensitivity of the bacterial cells to the replication inhibitor hydroxyurea. They identified a mutation in the gene encoding ObgE, a GTPase (guanosine triphosphatase) related to Ras. ObgE is a member of a family of GTPases that are present in eubacteria and eukaryotes that have been ascribed a broad range of possible functions in various organisms, including sporulation, vegetative growth, and stress responses. In E. coli , obgE is an essential gene, but it appears also to have a function that may serve as part of a DNA damage checkpoint. Genetic interactions of obgE mutants with other mutants in genes whose products function in DNA repair supported such a role. A mutation designed to inhibit GTPase activity of ObgE did inhibit its function, but whether GTP- and GDP-bound forms of the protein serve distinct roles and whether its activity is regulated by other factors (as the eukaryotic GTPases are) remain to be explored. J. J. Foti, J. Schienda, V. A. Sutera, S. T. Lovett, A bacterial G protein-mediated response to replication arrest. Mol. Cell 17 , 549-560 (2005). [PubMed]

Pregnancy enhances G protein activation and nitric oxide release from uterine arteries

We hypothesized that pregnancy modulates receptor-mediated responses of the uterine artery (UA) by altering G protein activation or coupling. Relaxation and contraction to NaF (0.5-11.5 mM), acetylcholine (10(-9)-10(-5) M), and bradykinin (10(-12)-3 x 10(-5) M) were measured in isolated UA of pregnant and nonpregnant guinea pigs. Responses were measured in the presence and absence of either cholera toxin (2 microg/ml) or pertussis toxin (Galpha(s) and Galpha(i) inhibitors, respectively). NaF relaxation was endothelium dependent and nitro-L-arginine sensitive (a nitric oxide synthase inhibitor). Relaxation to NaF, acetylcholine, and bradykinin were potentiated by pregnancy. Cholera but not pertussis toxin increased relaxation to acetylcholine and bradykinin in UA from nonpregnant animals, had no effect in UA from pregnant animals, and abolished the pregnancy-induced differences in acetylcholine relaxation. Cholera toxin potentiated the bradykinin-induced contraction of UA of both pregnant and nonpregnant animals, whereas pertussis toxin inhibited contraction of UA from pregnant animals only. Therefore, pregnancy may enhance agonist-stimulated endothelium-dependent relaxation and bradykinin-induced contraction of UA by inhibiting GTPase activity or enhancing Galpha(s) but not Galpha(i) activation in pregnant animals. Thus the diverse effects of pregnancy on UA responsiveness may result from hormonal modulation of G proteins coupled to their specific receptors.

Activatory effect of regucalcin on GTPase activity in rat liver plasma membranes.

The effect of regucalcin, a regulatory protein of Ca2+ signaling, on guanosine-5'-triphosphatase (GTPase) activity in isolated rat liver plasma membranes was investigated. GTPase activity was significantly increased by the addition of Ca2+ (25-100 microM) in the enzyme reaction mixture. Such an increase was not seen by other metals (Mg, Co, Zn, Cu, Ni and Mn) with 50 microM. The activatory effect of calcium (50 microM) was significantly decreased by calmodulin (2.5 and 5 microg/ml), indicating that it does not depend on calmodulin. The presence of regucalcin (0.1-0.5 microM) in the enzyme reaction mixture caused a significant increase in GTPase activity. This increase was not significantly enhanced by calcium (50 microM). GTPase activity was significantly increased by dithiothreitol (DTT; 5 mM), a protecting reagent of thiol (SH)-groups, while it was decreased by N-ethylmaleimide (NEM; 5 mM), a modifying reagent of SH-groups. The effect of calcium or regucalcin in increasing GTPase activity was not seen in the presence of NEM. Also, the activatory effect of calcium or regucalcin on GTPase was not seen in the presence of vanadate, an inhibitor of protein phosphorylation, which could inhibit GTPase activity. Moreover, the effect of regucalcin was not seen in the presence of digitonin (0.01%), a solubilizing reagent of membranous lipids, while the effect of calcium was not inhibited by digitonin. The present study demonstrates that regucalcin has an activatory effect on GTPase activity independently of Ca2+ in rat liver plasma membranes.