Analysis of Sex-Specific Prostanoid Production Using a Mouse Model of Selective Cyclooxygenase-2 Inhibition.

Background: Prostanoids are a family of lipid mediators formed from arachidonic acid by cyclooxygenase enzymes and serve as biomarkers of vascular function. Prostanoid production may be different in males and females indicating that different therapeutic approaches may be required during disease. Objectives: We examined sex-dependent differences in COX-related metabolites in genetically modified mice that produce a cyclooxygenase-2 (COX2) enzyme containing a tyrosine 385 to phenylalanine (Y385F) mutation. This mutation renders the COX2 enzyme unable to form a key intermediate radical required for complete arachidonic acid metabolism and provides a model of selective COX2 inhibition. Design and Methods: Mice heterozygous for the Y385F mutation in COX2 were mated to produce cohorts of wild-type, heterozygous, and COX2 mutant mice. We investigated whether the genotype distribution followed Mendelian genetics and studied whether sex-specific differences could be found in certain prostanoid levels measured in peritoneal macrophages and in urinary samples. Results: The inheritance of the COX2 mutation displayed a significant deviation with respect to Mendel's laws of genetics, with a lower-than-expected progeny of weaned COX2 mutant pups. In macrophages, prostaglandin E2 (PGE2) production following lipopolysaccharide (LPS) and interferon gamma (IFNγ) stimulation was COX2-dependent in both males and females, and data indicated that crosstalk between the nitric oxide (NO) and COX2 pathways may be sex specific. We observed significant differences in urinary PGE2 production by male and female COX2 mutant mice, with the loss of COX2 activity in male mice decreasing their ability to produce urinary PGE2. Finally, female mice across all 3 genotypes produced similar levels of urinary thromboxane (measured as 11-dehydro TxB2) at significantly higher levels than males, indicating a sex-related difference that is likely COX1-derived. Conclusions: Our findings clearly demonstrate that sex-related differences in COX-derived metabolites can be observed, and that other pathways (such as the NO pathway) are affected.

Structural basis and mechanism of activation of two different families of G proteins by the same GPCR.

The ß1-adrenergic receptor (ß1-AR) can activate two families of G proteins. When coupled to Gs, ß1-AR increases cardiac output, and coupling to Gi leads to decreased responsiveness in myocardial infarction. By comparative structural analysis of turkey ß1-AR complexed with either Gi or Gs, we investigate how a single G-protein-coupled receptor simultaneously signals through two G proteins. We find that, although the critical receptor-interacting C-terminal α5-helices on Gαi and Gαs interact similarly with ß1-AR, the overall interacting modes between ß1-AR and G proteins vary substantially. Functional studies reveal the importance of the differing interactions and provide evidence that the activation efficacy of G proteins by ß1-AR is determined by the entire three-dimensional interaction surface, including intracellular loops 2 and 4 (ICL2 and ICL4).

Structural Basis of the Activation of Heterotrimeric Gs-Protein by Isoproterenol-Bound ß1-Adrenergic Receptor.

Cardiac disease remains the leading cause of morbidity and mortality worldwide. The ß1-adrenergic receptor (ß1-AR) is a major regulator of cardiac functions and is downregulated in the majority of heart failure cases. A key physiological process is the activation of heterotrimeric G-protein Gs by ß1-ARs, leading to increased heart rate and contractility. Here, we use cryo-electron microscopy and functional studies to investigate the molecular mechanism by which ß1-AR activates Gs. We find that the tilting of α5-helix breaks a hydrogen bond between the sidechain of His373 in the C-terminal α5-helix and the backbone carbonyl of Arg38 in the N-terminal αN-helix of Gαs. Together with the disruption of another interacting network involving Gln59 in the α1-helix, Ala352 in the ß6-α5 loop, and Thr355 in the α5-helix, these conformational changes might lead to the deformation of the GDP-binding pocket. Our data provide molecular insights into the activation of G-proteins by G-protein-coupled receptors.

Structural Basis of Protein Arginine Methyltransferase Activation by a Catalytically Dead Homolog (Prozyme).

Prozymes are pseudoenzymes that stimulate the function of weakly active enzymes through complex formation. The major Trypanosoma brucei protein arginine methyltransferase, TbPRMT1 enzyme (ENZ), requires TbPRMT1 prozyme (PRO) to form an active heterotetrameric complex. Here, we present the X-ray crystal structure of the TbPRMT1 ENZ-Δ52PRO tetrameric complex with the cofactor product S-adenosyl-l-homocysteine (AdoHcy) at 2.4 Å resolution. The individual ENZ and PRO units adopt the highly-conserved PRMT domain architecture and form an antiparallel heterodimer that corresponds to the canonical homodimer observed in all previously reported PRMTs. In turn, two such heterodimers assemble into a tetramer both in the crystal and in solution with twofold rotational symmetry. ENZ is unstable in absence of PRO and incapable of forming a homodimer due to a steric clash of an ENZ-specific tyrosine within the dimerization arm, rationalizing why PRO is required to complement ENZ to form a PRMT dimer that is necessary, but not sufficient for PRMT activity. The PRO structure deviates from other, active PRMTs in that it lacks the conserved η2 310-helix within the Rossmann fold, abolishing cofactor binding. In addition to its chaperone function for ENZ, PRO substantially contributes to substrate binding. Heterotetramerization is required for catalysis, as heterodimeric ENZ-PRO mutants lack binding affinity and methyltransferase activity toward the substrate protein TbRGG1. Together, we provide a structural basis for TbPRMT1 ENZ activation by PRO heterotetramer formation, which is conserved across all kinetoplastids, and describe a chaperone function of the TbPRMT1 prozyme, which represents a novel mode of PRMT regulation.

Reducing cryoEM file storage using lossy image formats.

Recent advances in instrumentation and software for cryoEM have increased the applicability and utility of this method. High levels of automation and faster data acquisition rates require hard decisions to be made regarding data retention. Here we investigate the efficacy of data compression applied to aligned summed movie files. Surprisingly, these images can be compressed using a standard lossy method that reduces file storage by 90-95% and yet can still be processed to provide sub-2 Šreconstructed maps. We do not advocate this as an archival method, but it may provide a useful means for retaining images as an historical record, especially at large facilities.

High resolution single particle cryo-electron microscopy using beam-image shift.

Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (±0.1 µm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam-image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.

Benchmarking cryo-EM Single Particle Analysis Workflow.

Cryo electron microscopy facilities running multiple instruments and serving users with varying skill levels need a robust and reliable method for benchmarking both the hardware and software components of their single particle analysis workflow. The workflow is complex, with many bottlenecks existing at the specimen preparation, data collection and image analysis steps; the samples and grid preparation can be of unpredictable quality, there are many different protocols for microscope and camera settings, and there is a myriad of software programs for analysis that can depend on dozens of settings chosen by the user. For this reason, we believe it is important to benchmark the entire workflow, using a standard sample and standard operating procedures, on a regular basis. This provides confidence that all aspects of the pipeline are capable of producing maps to high resolution. Here we describe benchmarking procedures using a test sample, rabbit muscle aldolase.

Eicosapentaenoic Acid Modulates Trichomonas vaginalis Activity.

Trichomonas vaginalis is a sexually transmitted parasite and, while it is often asymptomatic in males, the parasite is associated with disease in both sexes. Metronidazole is an effective treatment for trichomoniasis, but resistant strains have evolved and, thus, it has become necessary to investigate other possible therapies. In this study, we examined the effects of native and oxidized forms of the sodium salts of eicosapentaenoic, docosahexaenoic, and arachidonic acids on T. vaginalis activity. Eicosapentaenoic acid was the most toxic with 190 and 380 µM causing approximately 90% cell death in Casu2 and ATCC 50142 strains, respectively. In contrast, oxidized eicosapentaenoic acid was the least toxic, requiring > 3 mM to inhibit activity, while low levels (10 µM) were associated with increased parasite density. Mass spectrometric analysis of oxidized eicosapentaenoic acid revealed C20 products containing one to six additional oxygen atoms and various degrees of bond saturation. These results indicate that eicosapentaenoic acid has different effects on T. vaginalis survival, depending on whether it is present in the native or oxidized form. A better understanding of lipid metabolism in T. vaginalis may facilitate the design of synthetic fatty acids that are effective for the treatment of metronidazole-resistant T. vaginalis.

Mass spectrometric analysis of oxidized eicosapentaenoic Acid sodium salt.

Eicosapentaenoic acid (EPA) is an omega-3 polyunsaturated fatty acid (PUFA) with 20 carbon atoms and 5 carbon-carbon double bonds. Mammalian cells cannot synthesize long chain PUFAs such as EPA de novo, and, thus, the most effective way to enrich cells in EPA is by dietary intake of fish oils. EPA supplementation causes an increase in its concentration in plasma lipids and in cell membrane phospholipids. Many beneficial effects of EPA supplementation have been noted, including (1) the potential to sensitize cancerous tumors towards chemotherapy, (2) the promotion of cardiovascular health, and (3) the alleviation of some mental disorders, but results from clinical trials have sometimes been disparate. In this study, we report the use of mass spectrometry to investigate the autoxidation of EPA, thereby demonstrating the formation of a variety of oxidized products. The oxidative stress of the patient may affect the response to EPA and may, in part, explain divergent results from clinical trials.