TRIM14 Is a Key Regulator of the Type I IFN Response during Mycobacterium tuberculosis Infection.

Tripartite motif-containing proteins (TRIMs) play a variety of recently described roles in innate immunity. Although many TRIMs regulate type I IFN expression following cytosolic nucleic acid sensing of viruses, their contribution to innate immune signaling and gene expression during bacterial infection remains largely unknown. Because Mycobacterium tuberculosis is an activator of cGAS-dependent cytosolic DNA sensing, we set out to investigate a role for TRIM proteins in regulating macrophage responses to M. tuberculosis In this study, we demonstrate that TRIM14, a noncanonical TRIM that lacks an E3 ubiquitin ligase RING domain, is a critical negative regulator of the type I IFN response in Mus musculus macrophages. We show that TRIM14 interacts with both cGAS and TBK1 and that macrophages lacking TRIM14 dramatically hyperinduce IFN stimulated gene (ISG) expression following M. tuberculosis infection, cytosolic nucleic acid transfection, and IFN-ß treatment. Consistent with a defect in resolution of the type I IFN response, Trim14 knockout macrophages have more phospho-Ser754 STAT3 relative to phospho-Ser727 and fail to upregulate the STAT3 target Socs3, which is required to turn off IFNAR signaling. These data support a model whereby TRIM14 acts as a scaffold between TBK1 and STAT3 to promote phosphorylation of STAT3 at Ser727 and resolve ISG expression. Remarkably, Trim14 knockout macrophages hyperinduce expression of antimicrobial genes like Nos2 and are significantly better than control cells at limiting M. tuberculosis replication. Collectively, these data reveal an unappreciated role for TRIM14 in resolving type I IFN responses and controlling M. tuberculosis infection.

Transcriptional regulation of human cyclic GMP-AMP synthase gene.

Cyclic GMP-AMP synthase (cGAS, cGAMP synthase) plays crucial roles in autoimmune disease, anti-tumor response, anti-senescence and anti-inflammatory response. Many studies have focused on cGAS-mediated signaling pathway. However, transcriptional mechanisms of cGAS gene have remained largely unknown. Here, we cloned the cGAS promoter region and characterized the molecular mechanisms controlling the cGAS transcriptional activity. By a series of 5' deletion and promoter constructions, we showed that the region (-414 to +76 relatives to the transcription start site) was sufficient for promoter activity. Mutation of Sp1 and CREB binding sites in this promoter region led to an apparent reduction of the cGAS promoter activity. Overexpression of Sp1 and CREB could obviously enhance promoter activity, whereas knocking-down of endogenous Sp1 and CREB markedly restrained the cGAS promoter activity. Sp1 and CREB binding to the cGAS promoter region in vivo was verified by Chromatin immunoprecipitation assay. These results pointed out that transcription factors Sp1 and CREB regulate the transcription of the cGAS gene.

Development of human cGAS-specific small-molecule inhibitors for repression of dsDNA-triggered interferon expression.

Cyclic GMP-AMP synthase (cGAS) is the primary sensor for aberrant intracellular dsDNA producing the cyclic dinucleotide cGAMP, a second messenger initiating cytokine production in subsets of myeloid lineage cell types. Therefore, inhibition of the enzyme cGAS may act anti-inflammatory. Here we report the discovery of human-cGAS-specific small-molecule inhibitors by high-throughput screening and the targeted medicinal chemistry optimization for two molecular scaffolds. Lead compounds from one scaffold co-crystallize with human cGAS and occupy the ATP- and GTP-binding active site. The specificity and potency of these drug candidates is further documented in human myeloid cells including primary macrophages. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.

Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target.

The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells.

Jasmonic acid-amino acid conjugation enzyme assays.

Jasmonic acid (JA) is activated for signaling by its conjugation to isoleucine (Ile) through an amide linkage. The Arabidopsis thaliana JASMONIC ACID RESISTANT1 (JAR1) enzyme carries out this Mg-ATP-dependent reaction in two steps, adenylation of the free carboxyl of JA, followed by condensation of the activated group to Ile. This chapter details the protocols used to detect and quantify the enzymatic activity obtained from a glutathione-S-transferase:JAR1 fusion protein produced in Escherichia coli, including an isotope exchange assay for the adenylation step and assays for the complete reaction that involve the high-performance liquid chromatography quantitation of adenosine monophosphate, a stoichiometric by-product of the reaction, and detection of the conjugation product by thin-layer chromatography or gas -chromatography/mass spectrometry.

Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway.

The presence of DNA in the cytoplasm of mammalian cells is a danger signal that triggers host immune responses such as the production of type I interferons. Cytosolic DNA induces interferons through the production of cyclic guanosine monophosphate-adenosine monophosphate (cyclic GMP-AMP, or cGAMP), which binds to and activates the adaptor protein STING. Through biochemical fractionation and quantitative mass spectrometry, we identified a cGAMP synthase (cGAS), which belongs to the nucleotidyltransferase family. Overexpression of cGAS activated the transcription factor IRF3 and induced interferon-ß in a STING-dependent manner. Knockdown of cGAS inhibited IRF3 activation and interferon-ß induction by DNA transfection or DNA virus infection. cGAS bound to DNA in the cytoplasm and catalyzed cGAMP synthesis. These results indicate that cGAS is a cytosolic DNA sensor that induces interferons by producing the second messenger cGAMP.

Role of key residues at the flavin mononucleotide (FMN):adenylyltransferase catalytic site of the bifunctional riboflavin kinase/flavin adenine dinucleotide (FAD) Synthetase from Corynebacterium ammoniagenes.

In mammals and in yeast the conversion of Riboflavin (RF) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) is catalysed by the sequential action of two enzymes: an ATP:riboflavin kinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT). However, most prokaryotes depend on a single bifunctional enzyme, FAD synthetase (FADS), which folds into two modules: the C-terminal associated with RFK activity and the N-terminal associated with FMNAT activity. Sequence and structural analysis suggest that the 28-HxGH-31, 123-Gx(D/N)-125 and 161-xxSSTxxR-168 motifs from FADS must be involved in ATP stabilisation for the adenylylation of FMN, as well as in FAD stabilisation for FAD phyrophosphorolysis. Mutants were produced at these motifs in the Corynebacterium ammoniagenes FADS (CaFADS). Their effects on the kinetic parameters of CaFADS activities (RFK, FMNAT and FAD pyrophosphorilase), and on substrates and product binding properties indicate that H28, H31, N125 and S164 contribute to the geometry of the catalytically competent complexes at the FMNAT-module of CaFADS.

The Rev1 translesion synthesis polymerase has multiple distinct DNA binding modes.

Rev1 is a eukaryotic DNA polymerase of the Y family involved in translesion synthesis (TLS), a major damage tolerance pathway that allows DNA replication at damaged templates. Uniquely amongst the Y family polymerases, the N-terminal part of Rev1, dubbed the BRCA1 C-terminal homology (BRCT) region, includes a BRCT domain. While most BRCT domains mediate protein-protein interactions, Rev1 contains a predicted α-helix N-terminal to the BRCT domain and in human Replication Factor C (RFC) such a BRCT region endows the protein with DNA binding capacity. Here, we studied the DNA binding properties of yeast and mouse Rev1. Our results show that the BRCT region of Rev1 specifically binds to a 5' phosphorylated, recessed, primer-template junction. This DNA binding depends on the extra α-helix, N-terminal to the BRCT domain. Surprisingly, a stretch of 20 amino acids N-terminal to the predicted α-helix is also critical for high-affinity DNA binding. In addition to 5' primer-template junction binding, Rev1 efficiently binds to a recessed 3' primer-template junction. These dual DNA binding characteristics are discussed in view of the proposed recruitment of Rev1 by 5' primer-template junctions, downstream of stalled replication forks.

Archaeal RibL: a new FAD synthetase that is air sensitive.

FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. Analyses of archaeal genomes did not reveal the presence of genes encoding either group, yet the archaea contain FAD. Our recent identification of a CTP-dependent archaeal riboflavin kinase strongly indicated the presence of a monofunctional FAD synthetase. Here we report the identification and characterization of an archaeal FAD synthetase. Methanocaldococcus jannaschii gene MJ1179 encodes a protein that is classified in the nucleotidyl transferase protein family and was previously annotated as glycerol-3-phosphate cytidylyltransferase (GCT). The MJ1179 gene was cloned and its protein product heterologously expressed in Escherichia coli. The resulting enzyme catalyzes the adenylation of FMN with ATP to produce FAD and PP(i). The MJ1179-derived protein has been designated RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Aerobically isolated RibL is active only under reducing conditions. RibL was found to require divalent metals for activity, the best activity being observed with Co(2+), where the activity was 4 times greater than that with Mg(2+). Alkylation of the two conserved cysteines in the C-terminus of the protein resulted in complete inactivation. RibL was also found to catalyze cytidylation of FMN with CTP, making the modified FAD, flavin cytidine dinucleotide (FCD). Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Also in contrast to other FAD synthetases, PP(i) inhibits the activity of RibL.

Purification and molecular characterization of a novel diadenosine 5',5'''-P(1),P(4)-tetraphosphate phosphorylase from Mycobacterium tuberculosis H37Rv.

In this study, Rv2613c, a protein that is encoded by the open reading frame Rv2613c in Mycobacterium tuberculosis H37Rv, was expressed, purified, and characterized for the first time. The amino acid sequence of Rv2613c contained a histidine triad (HIT) motif consisting of H-phi-H-phi-H-phi-phi, where phi is a hydrophobic amino acid. This motif has been reported to be the characteristic feature of several diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap4A) hydrolases that catalyze Ap4A to adenosine 5'-triphosphate (ATP) and adenosine monophosphate (AMP) or 2 adenosine 5'-diphosphate (ADP). However, enzymatic activity analyses for Rv2613c revealed that Ap4A was converted to ATP and ADP, but not AMP, indicating that Rv2613c has Ap4A phosphorylase activity rather than Ap4A hydrolase activity. The Ap4A phosphorylase activity has been reported for proteins containing a characteristic H-X-H-X-Q-phi-phi motif. However, no such motif was found in Rv2613c. In addition, the amino acid sequence of Rv2613c was significantly shorter compared to other proteins with Ap4A phosphorylase activity, indicating that the primary structure of Rv2613c differs from those of previously reported Ap4A phosphorylases. Kinetic analysis revealed that the K(m) values for Ap4A and phosphate were 0.10 and 0.94mM, respectively. Some enzymatic properties of Rv2613c, such as optimum pH and temperature, and bivalent metal ion requirement, were similar to those of previously reported yeast Ap4A phosphorylases. Unlike yeast Ap4A phosphorylases, Rv2613c did not catalyze the reverse phosphorolysis reaction. Taken together, it is suggested that Rv2613c is a unique protein, which has Ap4A phosphorylase activity with an HIT motif.

Functional analysis of the Burkholderia cenocepacia J2315 BceAJ protein with phosphomannose isomerase and GDP-D-mannose pyrophosphorylase activities.

The bceA(J) gene from the cystic fibrosis isolate Burkholderia cenocepacia J2315 encodes a 56-kDa bifunctional protein, with phosphomannose isomerase (PMI) and guanosine diphosphate (GDP)-mannose pyrophosphorylase (GMP) activities, a new member of the poorly characterised type II PMI class of proteins. Due to the lack of homology between the type II PMIs and the human PMI, this class of proteins are being regarded as interesting potential targets to develop new antimicrobials. The BceA(J) protein conserves the four typical motifs of type II PMIs: the pyrophosphorylase signature, the GMP active site, the PMI active site and the zinc-binding motif. After overproduction of BceA(J) by Escherichia coli as a histidine tag derivative, the protein was purified to homogeneity by affinity chromatography. The GMP activity is dependent on the presence of Mg(2+) or Ca(2+) as cofactors, while the PMI activity uses a broader range of divalent ions, in the order of activation Mg(2+) > Ca(2+) > Mn(2+) > Co(2+) > Ni(2+). The kinetic parameters K(m), V(max) and K(cat)/K(m) for the PMI and GMP activities were determined. Results suggest that the enzyme favours the formation of GDP-mannose instead of mannose catabolism, thus channelling precursors to the formation of glycoconjugates.

Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate.

BACKGROUND: We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP), in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. RESULTS: Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried out from thiamine diphosphate (ThDP) and ADP or ATP by a soluble high molecular mass nucleotidyl transferase. We partially purified this enzyme and characterized some of its functional properties. The enzyme activity had an absolute requirement for divalent metal ions, such as Mn2+ or Mg2+, as well as for a heat-stable soluble activator present in bacterial extracts. The enzyme has a pH optimum of 6.5-7.0 and a high Km for ThDP (5 mM), suggesting that, in vivo, the rate of AThTP synthesis is proportional to the free ThDP concentration. When ADP was used as the variable substrate at a fixed ThDP concentration, a sigmoid curve was obtained, with a Hill coefficient of 2.1 and an S0.5 value of 0.08 mM. The specificity of the AThTP synthesizing enzyme with respect to nucleotide substrate is restricted to ATP/ADP, and only ThDP can serve as the second substrate of the reaction. We tentatively named this enzyme ThDP adenylyl transferase (EC 2.7.7.65). CONCLUSION: This is the first demonstration of an enzyme activity transferring a nucleotidyl group on thiamine diphosphate to produce AThTP. The existence of a mechanism for the enzymatic synthesis of this compound is in agreement with the hypothesis of a non-cofactor role for thiamine derivatives in living cells.

In vitro analysis of the Escherichia coli AmtB-GlnK complex reveals a stoichiometric interaction and sensitivity to ATP and 2-oxoglutarate.

In Escherichia coli, the ammonia channel AmtB and the P(II) signal transduction protein GlnK constitute an ammonium sensory system that effectively couples the intracellular nitrogen regulation system to external changes in ammonium availability. Binding of GlnK to AmtB apparently inactivates the channel, thereby controlling ammonium influx in response to the intracellular nitrogen status. We designed an N-terminally histidine-tagged version of AmtB with a native C-terminal region in order to purify the AmtB-GlnK complex. Purification revealed a stable and direct interaction between AmtB and GlnK, thereby showing for the first time that stability of the complex does not require other proteins. The stoichiometry of the complex was determined by two independent approaches, both of which indicated a 1:1 ratio of AmtB to GlnK. We also showed by mass spectrometry that only the fully deuridylylated form of GlnK co-purifies with AmtB. The purified complex allowed in vitro studies of dissociation and association of AmtB and GlnK. The interaction of GlnK with AmtB is dependent on ATP and is also sensitive to 2-oxoglutarate. Our in vitro data suggest that in vivo association and dissociation of the complex might not only be dependent on the uridylylation status of GlnK but may also be influenced by intracellular pools of ATP and 2-oxoglutarate.

Hydrolytic approach for production of deoxyribonucleoside- and ribonucleoside-5 -monophosphates and enzymatic synthesis of their polyphosphates.

A new, simple, and ingenious method for enzymatic synthesis of deoxy- and ribonucleoside-5 -triphosphates (dNTP and NTP, respectively) has been developed. The method includes the following stages: hydrolysis of DNA with DNase and immobilized S1-nuclease, phosphorylation of the resulting deoxy- and ribonucleoside-5 -monophosphates (dNMP and NMP, respectively) with nucleotidyl kinase from Escherichia coli, and purification by chromatography of the synthesized dNTP and NTP. dNMP was phosphorylated using an ATP-regenerating system based on acetokinase from E. coli and lithium acetylphosphate.

Characterization of a thermophilic ATP-dependent DNA ligase from the euryarchaeon Pyrococcus horikoshii.

Archaea encode a DNA ligase composed of a C-terminal catalytic domain typical of ATP-dependent ligases plus an N-terminal domain similar to that found in eukaryotic cellular and poxvirus DNA ligases. All archaeal DNA ligases characterized to date have ATP-dependent adenylyltransferase and nick-joining activities. However, recent reports of dual-specificity ATP/NAD+ ligases in two Thermococcus species and Pyrococcus abyssi and an ATP/ADP ligase in Aeropyrum pernix raise the prospect that certain archaeal enzymes might exemplify an undifferentiated ancestral stage in the evolution of ligase substrate specificity. Here we analyze the biochemical properties of Pyrococcus horikoshii DNA ligase. P. horikoshii ligase catalyzes auto-adenylylation and nick sealing in the presence of a divalent cation and ATP; it is unable to utilize NAD+ or ADP to promote ligation in lieu of ATP. P. horikoshii ligase is thermophilic in vitro, with optimal adenylyltransferase activity at 90 degrees C and nick-joining activity at 70 to 90 degrees C. P. horikoshii ligase resembles the ligases of Methanobacterium thermautotrophicum and Sulfolobus shibatae in its strict specificity for ATP.

Cloning, sequencing, expression, and single-step purification of the adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU) from Salmonella typhimurium ATCC 19585.

The bi-functional enzyme, adenosylcobinamide kinase/adenosylcobinamide-phosphate guanylyltransferase (CobU), is involved in the biosynthesis of cobalamin in Salmonella typhimurium, and, therefore, can be used for the in vitro synthesis of analogs of B(12). Previously, five different steps were required to purify the recombinant enzyme from Escherichia coli. Here, we describe the cloning, sequencing, and expression of the cobU gene from S. typhimurium ATCC 19585 and, without introducing a purification tag sequence to the N- or C-terminus of the recombinant enzyme, a new single-step purification method based on hydrophobic interaction chromatography.

Purification and characterization of adenosine diphosphate glucose pyrophosphorylase from maize/potato mosaics.

Adenosine diphosphate glucose pyrophosphorylase (AGPase) catalyzes a rate-limiting step in starch biosynthesis. The reaction produces ADP-glucose and pyrophosphate from glucose-1-P and ATP. Investigations from a number of laboratories have shown that alterations in allosteric properties as well as heat stability of this enzyme have dramatic positive effects on starch synthesis in the potato (Solanum tuberosum) tuber and seeds of important cereals. Here, we report the characterization of purified recombinant mosaic AGPases derived from protein motifs normally expressed in the maize (Zea mays) endosperm and the potato tuber. These exhibit properties that should be advantageous when expressed in plants. We also present an in-depth characterization of the kinetic and allosteric properties of these purified recombinant AGPases. These data point to previously unrecognized roles for known allosteric effectors.

Allosteric regulation of the higher plant ADP-glucose pyrophosphorylase is a product of synergy between the two subunits.

The higher plant ADP-glucose pyrophosphorylase (AGPase) is a heterotetramer consisting of two regulatory large subunits (LSs) and two catalytic small subunits (SSs). To further characterize the roles of these subunits in determining enzyme function, different combinations of wildtype LS (LWT) and variant forms (LUpReg1, LM345) were co-expressed with wildtype SS (SWT) and variant forms (STG-15 and Sdevo330) and their enzyme properties compared to those measured for the heterotetrameric wildtype enzyme and SS homotetrameric enzymes. Analysis of the allosteric regulatory properties of the various enzymes indicates that although the LS is required for optimal activation by 3-phosphoglyceric acid and resistance to Pi, the overall allosteric regulatory and kinetic properties are specified by both subunits. Our results show that the regulatory and kinetic properties of AGPase are not simply due to the LS modulating the properties of the SS but, instead, are a product of synergistic interaction between the two subunits.

Rapid purification of the potato ADP-glucose pyrophosphorylase by polyhistidine-mediated chromatography.

In an attempt to obtain facile methods to purify the heterotetrameric ADP-glucose pyrophosphorylase (AGPase), polyhistidine tags were attached to either the large (LS) or small (SS) subunits of this oligomeric enzyme. The addition of polyhistidine tag to the N-terminus of the LS or SS and co-expression with its unmodified counterpart subunit resulted in substantial induction of enzyme activity. In contrast, attachment of a polyhistidine-containing peptide through the use of a commercially available pET vector or addition of polyhistidine tags to the C-terminal ends of either subunit resulted in poor expression and/or production of enzyme activity. Preliminary experiment showed that these polyhistidine N-terminal-tagged enzymes interacted with Ni-NTA-agarose, indicating that immobilized metal affinity chromatography (IMAC) would be useful for efficient purification of the heterotetrameric AGPases. When ion-exchange chromatography step was employed prior to the IMAC, the polyhistidine-tagged AGPases were purified to near homogeneity. Comparison of kinetic parameters between AGPases with and without the polyhistidine tags revealed that attachment of the polyhistidine did not alter the allosteric and catalytic properties of the enzymes. These results indicate that polyhistidine tags will be useful for the rapid purification of preparative amounts of AGPases for biochemical and physical studies.

The ADP-glucose pyrophosphorylase from Escherichia coli comprises two tightly bound distinct domains.

Computational analysis of ADP-glucose pyrophosphorylases predicts a fold with two domains. Co-expression of two polypeptides comprising residues 1-323 and 328-431 from the Escherichia coli ADP-glucose pyrophosphorylase yielded an enzyme form as active as the wild type. The only difference from the wild type was a slightly modified affinity for allosteric effectors. The two polypeptides could not be separated by chromatographic procedures. Separate expression of these polypeptides produced inactive unstable forms. All these results indicated that the ADP-glucose pyrophosphorylase comprises two domains with a strong interaction between them. That interaction is important for allosteric properties and structural stability.