HIF-1α Regulation of Cytokine Production following TLR3 Engagement in Murine Bone Marrow-Derived Macrophages Is Dependent on Viral Nucleic Acid Length and Glucose Availability.

Hypoxia-inducible factor-1α (HIF-1α) is an important regulator of glucose metabolism and inflammatory cytokine production in innate immune responses. Viruses modulate HIF-1α to support viral replication and the survival of infected cells, but it is unclear if this transcription factor also plays an important role in regulating antiviral immune responses. In this study, we found that short and long dsRNA differentially engage TLR3, inducing distinct levels of proinflammatory cytokine production (TNF-α and IL-6) in bone marrow-derived macrophages from C57BL/6 mice. These responses are associated with differential accumulation of HIF-1α, which augments NF-κB activation. Unlike TLR4 responses, increased HIF-1α following TLR3 engagement is not associated with significant alterations in glycolytic activity and was more pronounced in low glucose conditions. We also show that the mechanisms supporting HIF-1α stabilization may differ following stimulation with short versus long dsRNA and that pyruvate kinase M2 and mitochondrial reactive oxygen species play a central role in these processes. Collectively, this work suggests that HIF-1α may fine-tune proinflammatory cytokine production during early antiviral immune responses, particularly when there is limited glucose availability or under other conditions of stress. Our findings also suggest we may be able to regulate the magnitude of proinflammatory cytokine production during antiviral responses by targeting proteins or molecules that contribute to HIF-1α stabilization.

Differential remodeling of the electron transport chain is required to support TLR3 and TLR4 signaling and cytokine production in macrophages.

Increasing evidence suggests that mitochondria play a critical role in driving innate immune responses against bacteria and viruses. However, it is unclear if differential reprogramming of mitochondrial function contributes to the fine tuning of pathogen specific immune responses. Here, we found that TLR3 and TLR4 engagement on murine bone marrow derived macrophages was associated with differential remodeling of electron transport chain complex expression. This remodeling was associated with differential accumulation of mitochondrial and cytosolic ROS, which were required to support ligand specific inflammatory and antiviral cytokine production. We also found that the magnitude of TLR3, but not TLR4, responses were modulated by glucose availability. Under conditions of low glucose, TLR3 engagement was associated with increased ETC complex III expression, increased mitochondrial and cytosolic ROS and increased inflammatory and antiviral cytokine production. This amplification was selectively reversed by targeting superoxide production from the outer Q-binding site of the ETC complex III. These results suggest that ligand specific modulation of the ETC may act as a rheostat that fine tunes innate immune responses via mitochondrial ROS production. Modulation of these processes may represent a novel mechanism to modulate the nature as well as the magnitude of antiviral vs. inflammatory immune responses.

Transcriptional Profiling Suggests Extensive Metabolic Rewiring of Human and Mouse Macrophages during Early Interferon Alpha Responses.

Emerging evidence suggests that cellular metabolism plays a critical role in regulating immune activation. Alterations in energy and lipid and amino acid metabolism have been shown to contribute to type I interferon (IFN) responses in macrophages, but the relationship between metabolic reprogramming and the establishment of early antiviral function remains poorly defined. Here, we used transcriptional profiling datasets to develop global metabolic signatures associated with early IFN-α responses in two primary macrophage model systems: mouse bone marrow-derived macrophages (BMM) and human monocyte-derived macrophages (MDM). Short-term stimulation with IFN-α (<4 hours) was associated with significant metabolic rewiring, with >500 metabolic genes altered in mouse and human macrophage models. Pathway and network analysis identified alterations in genes associated with cellular bioenergetics, cellular oxidant status, cAMP/AMP and cGMP/GMP ratios, branched chain amino acid catabolism, cell membrane composition, fatty acid synthesis, and ß-oxidation as key features of early IFN-α responses. These changes may have important implications for initial establishment of antiviral function in these cells.

Development of polymorphic microsatellite markers for Indian tobacco, Lobelia inflata (Campanulaceae)(1.).

PREMISE OF THE STUDY: Nuclear microsatellite markers were developed for Lobelia inflata (Campanulaceae), an obligately self-fertilizing plant species, for use in the study of temporal fluctuation in allele frequency and of the genetic structure within and among populations. • METHODS AND RESULTS: We developed 28 primer pairs for L. inflata, all of which amplify CT dinucleotide repeats. We evaluated amplification of these loci in 53 L. inflata individuals at three sites in eastern North America and found that 24 loci showed microsatellite polymorphism. We also found that 16 loci amplified successfully in L. cardinalis, and 11 amplified successfully in L. siphilitica. • CONCLUSIONS: These primers will be useful for assessing allelic diversity within and among populations of L. inflata, and show potential for use in congeneric species.

Expression and characterization of the Arabidopsis thaliana 11S globulin family.

The 11S globulins are the principal seed storage proteins in a variety of major crop species, including members of the legume and mustard families. They are targets for protein engineering studies attempting to alter the physicochemical properties of seed protein extracts (e.g. soybean) and to improve the nutritional quality of important agricultural crops. A key factor that has limited the success of this approach to date is insufficient accumulation of the engineered protein variants in vivo due to their improper folding and/or reduced stability, compared to the native protein. We have developed the Arabidopsis thaliana 11S proglobulins as a model system to enable studies exploring the factors underlying structural stability in this family of proteins. Yields of 1.5-4 mg/L were achieved for the three A. thaliana 11S proglobulins expressed in the Origami Escherichia coli cell line in super broth media at 20°C for 16 h and purified via immobilized-metal affinity chromatography. We also demonstrate that differential scanning fluorimetry is an effective and accessible technique to facilitate the screening of variants to enable the successful engineering of 11S seed storage proteins. The relative in vitro stability of the A. thaliana 11S proglobulins (proAtCRU1>proAtCRU3>proAtCRU2) is consistent between chemical and thermal denaturation studies.

Exploration of the six tryptophan residues of Escherichia coli cystathionine ß-lyase as probes of enzyme conformational change.

Cystathionine ß-lyase (CBL) catalyzes the hydrolysis of l-cystathionine (l-Cth), producing l-homocysteine (l-Hcys), pyruvate and ammonia, in the second step of the transsulfuration pathway of bacteria and plants. A series of 17 site-directed variants of Escherichia coli CBL (eCBL) was constructed to probe the contributions of the six tryptophan residues (W131, W188, W230, W276, W300 and W340) to the fluorescence spectrum of eCBL and to assess their mutability and utility as conformational probes. The effects of these Trp→Phe substitutions on kcat and Km(l)(-Cth) are less than 2-fold, with the exception of the 8-fold increase in Km(l)(-Cth) observed for eCBL-W340F. The midpoint of thermal denaturation, as monitored by circular dichroism spectroscopy, is reduced 4.7°C by the W188F substitution while the targeted replacement of the other five tryptophans alter Tm by less than 1.7°C. The fluorescence spectrum of eCBL is dominated by W230 and the contribution of W340, situated in the active site, is minor. The observed 5-fold increase in the 336 nm fluorescence emission of W188 between 0 and 2M urea, suggests a conformational change at the domain interface. Residues W188 and W340, conserved in proteobacterial CBL enzymes, are situated at the core of the domain interface that forms the active-site cleft. The results of this study suggest that W188 is a useful probe of subtle conformational changes at the domain interface and active site.

Exploration of structure-function relationships in Escherichia coli cystathionine γ-synthase and cystathionine ß-lyase via chimeric constructs and site-specific substitutions.

Cystathionine γ-synthase (CGS) and cystathionine ß-lyase (CBL) share a common structure and several active-site residues, but catalyze distinct side-chain rearrangements in the two-step transsulfuration pathway that converts cysteine to homocysteine, the precursor of methionine. A series of 12 chimeric variants of Escherichia coli CGS (eCGS) and CBL (eCBL) was constructed to probe the roles of two structurally distinct, ~25-residue segments situated in proximity to the amino and carboxy termini and located at the entrance of the active-site. In vivo complementation of methionine-auxotrophic E. coli strains, lacking the genes encoding eCGS and eCBL, demonstrated that exchange of the targeted regions impairs the activity of the resulting enzymes, but does not produce a corresponding interchange of reaction specificity. In keeping with the in vivo results, the catalytic efficiency of the native reactions is reduced by at least 95-fold, and α,ß versus α,γ-elimination specificity is not modified. The midpoint of thermal denaturation monitored by circular dichroism, ranges between 59 and 80°C, compared to 66°C for the two wild-type enzymes, indicating that the chimeric enzymes adopt a stable folded structure and that the observed reductions in catalytic efficiency are due to reorganization of the active site. Alanine-substitution variants of residues S32 and S33, as well as K42 of eCBL, situated in proximity to and within, respectively, the targeted amino-terminal region were also investigated to explore their role as determinants of reaction specificity via positioning of key active-site residues. The catalytic efficiency of the S32A, S33A and the K42A site-directed variants of eCBL is reduced by less than 10-fold, demonstrating that, while these residues may participate in positioning S339, which tethers the catalytic base, their role is minor.

Identification of cystathionine γ-synthase and threonine synthase from Cicer arietinum and Lens culinaris.

In plants, cystathionine γ-synthase (CGS) and threonine synthase (TS) compete for the branch-point metabolite O-phospho-L-homoserine. These enzymes are potential targets for metabolic engineering studies, aiming to alter the flux through the competing methionine and threonine biosynthetic pathways, with the goal of increasing methionine production. Although CGS and TS have been characterized in the model organisms Escherichia coli and Arabidopsis thaliana, little information is available on these enzymes in other, particularly plant, species. The functional CGS and TS coding sequences from the grain legumes Cicer arietinum (chickpea) and Lens culinaris (lentil) identified in this study share approximately 80% amino acid sequence identity with the corresponding sequences from Glycine max. At least 7 active-site residues of grain legume CGS and TS are conserved in the model bacterial enzymes, including the catalytic base. Putative processing sites that remove the targeting sequence and result in functional TS were identified in the target species.

Exploration of the active site of Escherichia coli cystathionine γ-synthase.

Cystathionine γ-synthase (CGS) catalyzes the condensation of O-succinyl-L-homoserine (L-OSHS) and L-cysteine (L-Cys), to produce L-cystathionine (L-Cth) and succinate, in the first step of the bacterial transsulfuration pathway. In the absence of L-Cys, the enzyme catalyzes the futile α,γ-elimination of L-OSHS, yielding succinate, α-ketobutyrate, and ammonia. A series of 16 site-directed variants of Escherichia coli CGS (eCGS) was constructed to probe the roles of active-site residues D45, Y46, R48, R49, Y101, R106, N227, E325, S326, and R361. The effects of these substitutions on the catalytic efficiency of the α,γ-elimination reaction range from a reduction of only ∼2-fold for R49K and the E325A,Q variants to 310- and 760-fold for R361K and R48K, respectively. A similar trend is observed for the k(cat) /K(m)(l-OSHS) of the physiological, α,γ-replacement reaction. The results of this study suggest that the arginine residues at positions 48, 106 and 361 of eCGS, conserved in bacterial CGS sequences, tether the distal and α-carboxylate moieties, respectively, of the L-OSHS substrate. In contrast, with the exception of the 13-fold increase observed for R106A, the K(m)(l-Cys) is not markedly affected by the site-directed replacement of the residues investigated. The decrease in k(cat) observed for the S326A variant reflects the role of this residue in tethering the side chain of K198, the catalytic base. Although no structures exist of eCGS bound to active-site ligands, the roles of individual residues is consistent with the structures inhibitor complexes of related enzymes. Substitution of D45, E325, or Y101 enables a minor transamination activity for the substrate L-Ala.

Characterization of site-directed mutants of residues R58, R59, D116, W340 and R372 in the active site of E. coli cystathionine beta-lyase.

Cystathionine beta-lyase (CBL) catalyzes the hydrolysis of L-cystathionine (L-Cth) to produce L-homocysteine, pyruvate, and ammonia. A series of active-site mutants of Escherichia coli CBL (eCBL) was constructed to investigate the roles of residues R58, R59, D116, W340, and R372 in catalysis and inhibition by aminoethoxyvinylglycine (AVG). The effects of these mutations on the k(cat)/K(m) (L-Cth) for the beta-elimination reaction range from a reduction of only 3-fold for D116A and D116N to 6 orders of magnitude for the R372L and R372A mutants. The order of importance of these residues for the hydrolysis of L-Cth is: R372 >> R58 > W340 approximately R59 > D116. Comparison of the kinetic parameters for L-Cth hydrolysis with those for inhibition of eCBL by AVG demonstrates that residue R58 tethers the distal carboxylate group of the substrate and confirms that residues W340 and R372 interact with the alpha-carboxylate moiety. The increase in the pK(a) of the acidic limb and decrease in the pK(a) of the basic limb of the k(cat)/K(m) (L-Cth) versus pH profiles of the R58K and R58A mutants, respectively, support a role for this residue in modulating the pK(a) of an active-site residue.