Visualizing Cytoophidia Expression in Drosophila Follicle Cells via Immunohistochemistry.

We describe a user-friendly immunohistochemical approach for the detection of protein localization in Drosophila ovaries, here focusing on CTP synthase. This approach mainly uses fluorescently labeled antibodies to detect single, double, or multiple antigens. We provide a step-by-step protocol with detailed notes and tips, a simplified method that can also be adapted to detect protein localization beyond Drosophila ovaries.

Site-specific biotinylation of purified proteins using BirA.

The binding between biotin and streptavidin or avidin is one of the strongest known non-covalent biological interactions. The (strept)avidin-biotin interaction has been widely used for decades in biological research and biotechnology. Therefore labeling of purified proteins by biotin is a powerful way to achieve protein capture, immobilization, and functionalization, as well as multimerizing or bridging molecules. Chemical biotinylation often generates heterogeneous products, which may have impaired function. Enzymatic biotinylation with E. coli biotin ligase (BirA) is highly specific in covalently attaching biotin to the 15 amino acid AviTag peptide, giving a homogeneous product with high yield. AviTag can conveniently be added genetically at the N-terminus, C-terminus, or in exposed loops of a target protein. We describe here procedures for AviTag insertion by inverse PCR, purification of BirA fused to glutathione-S-transferase (GST-BirA) from E. coli, BirA biotinylation of purified protein, and gel-shift analysis by SDS-PAGE to quantify the extent of biotinylation.

Glycinamide ribonucleotide formyl transferase is frequently overexpressed in glioma and critically regulates the proliferation of glioma cells.

AIMS: Current treatments for the most common form of brain tumor, glioma, are disappointing in their effectiveness. Low expression levels of GART, an enzyme in the core nucleotide metabolism, significantly correlate with chemosensitivity, conferring a survival advantage to tumor cells. Our study aimed to explore the expression and function of GART in glioma. METHODS: Immunohistochemical and Western blot analysis were performed in 70 cases of human gliomas and normal brain tissues. We mainly used cell growth assay and multicellular tumor spheroid formation assay to evaluate the proliferation and chemosensitivity of glioma cells. RESULTS: High GART expression (most cancer cells cytoplasm stained) was observed in 70 specimens and was related to the grade of malignancy. We also reviewed each grade of tumors separately and investigated whether GART expression predicted patient survival within each subgroup. In brief, GART overexpression was significantly associated with overall survival (P=0.03). Interestingly, transfecting cells with GART-siRNA suppressed proliferation and enhanced temozolomide (TMZ)-induced apoptosis in glioma cells. CONCLUSION: The current results showed that GART expression was associated with glioma grade and that high GART protein expression might be related to poor outcome.

Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion.

Known proteins associated with the cell-adhesion protein E-cadherin include catenins and proteins involved in signaling, trafficking and actin organization. However, the list of identified adherens junction proteins is likely to be incomplete, limiting investigation into this essential cell structure. To expand the inventory of potentially relevant proteins, we expressed E-cadherin fused to biotin ligase in MDCK epithelial cells, and identified by mass spectrometry neighboring proteins that were biotinylated. The most abundant of the 303 proteins identified were catenins and nearly 40 others that had been previously reported to influence cadherin function. Many others could be rationalized as novel candidates for regulating the adherens junction, cytoskeleton, trafficking or signaling. We further characterized lipoma preferred partner (LPP), which is present at both cell contacts and focal adhesions. Knockdown of LPP demonstrated its requirement for E-cadherin-dependent adhesion and suggested that it plays a role in coordination of the cell-cell and cell-substrate cytoskeletal interactions. The analysis of LPP function demonstrates proof of principle that the proteomic analysis of E-cadherin proximal proteins expands the inventory of components and tools for understanding the function of E-cadherin.

Three promoters regulate the transcriptional activity of the human holocarboxylase synthetase gene.

Holocarboxylase synthetase (HLCS) is the only protein biotin ligase in the human proteome. HLCS-dependent biotinylation of carboxylases plays crucial roles in macronutrient metabolism. HLCS appears to be an essential part of multiprotein complexes in the chromatin that cause gene repression and contribute toward genome stability. Consistent with these essential functions, HLCS knockdown causes strong phenotypes including shortened life span and low stress resistance in Drosophila melanogaster, and de-repression of long-terminal repeats in humans, other mammalian cell lines and Drosophila. Despite previous observations that the expression of HLCS depends on biotin status in rats and in human cell lines, little is known about the regulation of HLCS expression. The goal of this study was to identify promoters that regulate the expression of the human HLCS gene. Initially, the human HLCS locus was interrogated in silico using predictors of promoters including sequences of HLCS mRNA and expressed sequence tags, CpG islands, histone marks denoting transcriptionally poised chromatin, transcription factor binding sites and DNaseI hypersensitive regions. Our predictions revealed three putative HLCS promoters, denoted P1, P2 and P3. Promoters lacked a TATA box, which is typical for housekeeping genes. When the three promoters were cloned into a luciferase reporter plasmid, reporter gene activity was at least three times background noise in human breast, colon and kidney cell lines; activities consistently followed the pattern P1>>P3>P2. Promoter activity depended on the concentration of biotin in culture media, but the effect was moderate. We conclude that we have identified promoters in the human HLCS gene.

Good codons, bad transcript: large reductions in gene expression and fitness arising from synonymous mutations in a key enzyme.

Biased codon usage in protein-coding genes is pervasive, whereby amino acids are largely encoded by a specific subset of possible codons. Within individual genes, codon bias is stronger at evolutionarily conserved residues, favoring codons recognized by abundant tRNAs. Although this observation suggests an overall pattern of selection for translation speed and/or accuracy, other work indicates that transcript structure or binding motifs drive codon usage. However, our understanding of codon bias evolution is constrained by limited experimental data on the fitness effects of altering codons in functional genes. To bridge this gap, we generated synonymous variants of a key enzyme-coding gene in Methylobacterium extorquens. We found that mutant gene expression, enzyme production, enzyme activity, and fitness were all significantly lower than wild-type. Surprisingly, encoding the gene using only rare codons decreased fitness by 40%, whereas an allele coded entirely by frequent codons decreased fitness by more than 90%. Increasing gene expression restored mutant fitness to varying degrees, demonstrating that the fitness disadvantage of synonymous mutants arose from a lack of beneficial protein rather than costs of protein production. Protein production was negatively correlated with the frequency of motifs with high affinity for the anti-Shine-Dalgarno sequence, suggesting ribosome pausing as the dominant cause of low mutant fitness. Together, our data support the idea that, although a particular set of codons are favored on average across a genome, in an individual gene selection can either act for or against codons depending on their local context.

Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity.

Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

Regulation of human cytidine triphosphate synthetase 2 by phosphorylation.

Cytidine triphosphate synthetase (CTPS) is the rate-limiting enzyme in de novo CTP synthesis and is required for the formation of RNA, DNA, and phospholipids. This study determined the kinetic properties of the individual human CTPS isozymes (hCTPS1 and hCTPS2) and regulation through substrate concentration, oligomerization, and phosphorylation. Kinetic analysis demonstrated that both hCTPS1 and hCTPS2 were maximally active at physiological concentrations of ATP, GTP, and glutamine, whereas the K(m) and IC(50) values for the substrate UTP and the product CTP, respectively, were close to their physiological concentrations, indicating that the intracellular concentrations of UTP and CTP may precisely regulate hCTPS activity. Low serum treatment increased hCTPS2 phosphorylation, and five probable phosphorylation sites were identified in the hCTPS2 C-terminal domain. Metabolic labeling of hCTPS2 with [(32)P]H(3)PO(4) demonstrated that Ser(568) and Ser(571) were two major phosphorylation sites, and additional studies demonstrated that Ser(568) was phosphorylated by casein kinase 1 both in vitro and in vivo. Interestingly, mutation of Ser(568) (S568A) but not Ser(571) significantly increased hCTPS2 activity, demonstrating that Ser(568) is a major inhibitory phosphorylation site. The S568A mutation had a greater effect on the glutamine than ammonia-dependent activity, indicating that phosphorylation of this site may influence the glutaminase domain of hCTPS2. Deletion of the C-terminal regulatory domain of hCTPS1 also greatly increased the V(max) of this enzyme. In summary, this is the first study to characterize the kinetic properties of hCTPS1 and hCTPS2 and to identify Ser(568) as a major site of CTPS2 regulation by phosphorylation.

Generation of peptide MHC class I monomers and multimers through ligand exchange.

The recognition of defined antigen-MHC complexes by antigen-specific T cells forms the molecular basis of T cell immunity. It has been shown that fluorescently labeled recombinant MHC tetramers can be utilized to detect antigen-specific T cells by flow cytometry. Since this first description, MHC tetramers and other types of MHC multimers have become a core tool to monitor the development of disease- and therapy-induced antigen-specific T cell responses both in humans and in animal model systems. This unit describes a set of protocols that transform classical MHC multimer technology into a high-throughput platform, allowing one to produce large collections of MHC class I molecules charged with different peptides. This technology is based on the development of conditional MHC ligands that can be triggered to self-destruct while in the MHC-bound state.

The 1.6 A crystal structure of Mycobacterium smegmatis MshC: the penultimate enzyme in the mycothiol biosynthetic pathway.

Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of GlcN-Ins and l-cysteine to form l-Cys-GlcN-Ins, the penultimate step in mycothiol biosynthesis. Attempts to crystallize the native, full-length MshC have been unsuccessful. However, incubation of the enzyme with the cysteinyl adenylate analogue, 5'-O-[N-(l-cysteinyl)-sulfamonyl]adenosine (CSA), followed by a 24-h limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. The three-dimensional structure of MshC with CSA bound in the active site was solved and refined to 1.6 A. The refined structure exhibited electron density corresponding to the entire 47 kDa MshC molecule, with the exception of the KMSKS loop (residues 285-297), a loop previously implicated in the formation of the adenylate in related tRNA synthases. The overall tertiary fold of MshC is similar to that of cysteinyl-tRNA synthetase, with a Rossmann fold catalytic domain. The interaction of the thiolate of CSA with a zinc ion at the base of the active site suggests that the metal ion participates in amino acid binding and discrimination. A number of active site residues were observed to interact with the ligand, suggesting a role in substrate binding and catalysis. Analysis utilizing modeling of the proteolyzed loop and GlcN-Ins docking, as well as the examination of sequence conservation in the active site suggests similarities and differences between cysteinyl-tRNA synthetases and MshC in recognition of the substrates for their respective reactions.

Impaired biotinidase activity disrupts holocarboxylase synthetase expression in late onset multiple carboxylase deficiency.

Biotinidase catalyzes the hydrolysis of the vitamin biotin from proteolytically degraded biotin-dependent carboxylases. This key reaction makes the biotin available for reutilization in the biotinylation of newly synthesized apocarboxylases. This latter reaction is catalyzed by holocarboxylase synthetase (HCS) via synthesis of 5'-biotinyl-AMP (B-AMP) from biotin and ATP, followed by transfer of the biotin to a specific lysine residue of the apocarboxylase substrate. In addition to carboxylase activation, B-AMP is also a key regulatory molecule in the transcription of genes encoding apocarboxylases and HCS itself. In humans, genetic deficiency of HCS or biotinidase results in the life-threatening disorder biotin-responsive multiple carboxylase deficiency, characterized by a reduction in the activities of all biotin-dependent carboxylases. Although the clinical manifestations of both disorders are similar, they differ in some unique neurological characteristics whose origin is not fully understood. In this study, we show that biotinidase deficiency not only reduces net carboxylase biotinylation, but it also impairs the expression of carboxylases and HCS by interfering with the B-AMP-dependent mechanism of transcription control. We propose that biotinidase-deficient patients may develop a secondary HCS deficiency disrupting the altruistic tissue-specific biotin allocation mechanism that protects brain metabolism during biotin starvation.

Heterologous protein production in Escherichia coli using the propionate-inducible pPro system by conventional and auto-induction methods.

We examined expression of two plant genes encoding coclaurine N-methyltransferase (CMT) and norcoclaurine synthase (NCS) in Escherichia coli from the Salmonella entericaprpBCDE promoter (P(prpB)) and compared it to that from the strongest IPTG-inducible promoter, P(T7). In contrast to our previous study showing slightly higher production of green fluorescent protein (GFP) from the pPro system compared to that from the T7 system, production of two plant proteins CMT and NCS from P(prpB) was 2- to 4-fold higher than that from P(T7). Unlike P(T7), expression from P(prpB) did not reduce cell growth even when highly induced, indicating that this propionate-inducible system is more efficient for overproduction of proteins that result in growth inhibition. In an auto-induction experiment, which does not require monitoring the culture or adding inducer during cell growth, the pPro system exhibited much higher protein production than the T7 system. These results strongly indicate that the pPro system is well-suited for overproduction of recombinant proteins.

Targeting and purification of metabolically biotinylated baculovirus.

Targeting viral entry is one of the major goals in the development of vectors for gene therapy. Ideally, the coupling of each new targeting motif would not require changes in vector structure. To achieve this, we developed novel metabolically biotinylated baculoviral vectors by displaying a small biotin acceptor peptide (BAP) fused either to different sites in the baculovirus glycoprotein gp64 or to the transmembrane anchor of vesicular stomatitis virus G protein. Baculoviral particles were biotinylated during vector production by coexpression of Escherichia coli biotin ligase (BirA). The insertion of BAP at amino acid position 283 of gp64 resulted in the most efficient biotin display. Unlike vectors with lower biotin display, these vectors also showed improved transduction when retargeted to transferrin, epidermal growth factor, and CD46 receptors overexpressed on rat glioma and human ovarian carcinoma cells. Biotinylated baculoviral vectors could also be concentrated by one-step magnetic particle-based capture to reach titers up to 10(10) plaque-forming units/ml. These results demonstrate the utility of metabolically biotinylated baculovirus for vector targeting and viral purification applications.

Cloning, expression, crystallization and preliminary X-ray data analysis of norcoclaurine synthase from Thalictrum flavum.

Norcoclaurine synthase (NCS) catalyzes the condensation of 3,4-dihydroxyphenylethylamine (dopamine) and 4-hydroxyphenylacetaldehyde (4-HPAA) as the first committed step in the biosynthesis of benzylisoquinoline alkaloids in plants. The protein was cloned, expressed and purified. Crystals were obtained at 294 K by the hanging-drop vapour-diffusion method using ammonium sulfate and sodium chloride as precipitant agents and diffract to better than 3.0 A resolution using a synchrotron-radiation source. The crystals belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 86.31, c = 118.36 A. A selenomethionine derivative was overexpressed, purified and crystallized in the same space group. A complete MAD data set was collected at 2.7 A resolution. The model is under construction.

Mechanistic studies on norcoclaurine synthase of benzylisoquinoline alkaloid biosynthesis: an enzymatic Pictet-Spengler reaction.

Norcoclaurine synthase catalyzes an asymmetric Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. This is the first committed step in the biosynthesis of the benzylisoquinoline alkaloids that include morphine and codeine. In this work, the gene encoding for the Thalictrum flavum norcoclaurine synthase is highly overexpressed in Escherichia coli and the resulting His-tagged recombinant enzyme is purified for the first time. A continuous assay based on circular dichroism spectroscopy is developed and used to monitor the kinetics of the enzymatic reaction. Dopamine analogues bearing a methoxy or hydrogen substituent in place of the C-1 phenolic group were readily accepted by the enzyme whereas those bearing the same substituents at C-2 were not. This supports a mechanism involving a two-step cyclization of the putative iminium ion intermediate that does not proceed via a spirocyclic intermediate. The reaction of [3,5,6-2H]dopamine was found to be slowed by a kinetic isotope effect of 1.7 +/- 0.1 on the value of kcat/KM. This is interpreted as showing that the deprotonation step causing rearomatization is partially rate determining in the overall reaction.

Regulation of human cytidine triphosphate synthetase 1 by glycogen synthase kinase 3.

Cytidine triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3'-monoxime and GSK3beta short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [(32)P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3'-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.

Engineering central metabolic pathways for high-level flavonoid production in Escherichia coli.

The identification of optimal genotypes that result in improved production of recombinant metabolites remains an engineering conundrum. In the present work, various strategies to reengineer central metabolism in Escherichia coli were explored for robust synthesis of flavanones, the common precursors of plant flavonoid secondary metabolites. Augmentation of the intracellular malonyl coenzyme A (malonyl-CoA) pool through the coordinated overexpression of four acetyl-CoA carboxylase (ACC) subunits from Photorhabdus luminescens (PlACC) under a constitutive promoter resulted in an increase in flavanone production up to 576%. Exploration of macromolecule complexes to optimize metabolic efficiency demonstrated that auxiliary expression of PlACC with biotin ligase from the same species (BirAPl) further elevated flavanone synthesis up to 1,166%. However, the coexpression of PlACC with Escherichia coli BirA (BirAEc) caused a marked decrease in flavanone production. Activity improvement was reconstituted with the coexpression of PlACC with a chimeric BirA consisting of the N terminus of BirAEc and the C terminus of BirAPl. In another approach, high levels of flavanone synthesis were achieved through the amplification of acetate assimilation pathways combined with the overexpression of ACC. Overall, the metabolic engineering of central metabolic pathways described in the present work increased the production of pinocembrin, naringenin, and eriodictyol in 36 h up to 1,379%, 183%, and 373%, respectively, over production with the strains expressing only the flavonoid pathway, which corresponded to 429 mg/liter, 119 mg/liter, and 52 mg/liter, respectively.

Expression, purification, characterization, and in vivo targeting of trypanosome CTP synthetase for treatment of African sleeping sickness.

African sleeping sickness is a fatal disease caused by two parasite subspecies: Trypanosoma brucei gambiense and T. b. rhodesiense. We previously reported that trypanosomes have extraordinary low CTP pools compared with mammalian cells. Trypanosomes also lack salvage of cytidine/cytosine making the parasite CTP synthetase a potential target for treatment of the disease. In this study, we have expressed and purified recombinant T. brucei CTP synthetase. The enzyme has a higher K(m) value for UTP than the mammalian CTP synthetase, which in combination with a lower UTP pool may account for the low CTP pool in trypanosomes. The activity of the trypanosome CTP synthetase is irreversibly inhibited by the glutamine analogue acivicin, a drug extensively tested as an antitumor agent. There is a rapid uptake of acivicin in mice both given intraperitoneally and orally by gavage. Daily injection of acivicin in trypanosome-infected mice suppressed the infection up to one month without any significant loss of weight. Experiments with cultured bloodstream T. brucei showed that acivicin is trypanocidal if present at 1 mum concentration for at least 4 days. Therefore, acivicin may qualify as a drug with "desirable" properties, i.e. cure within 7 days, according to the current Target Product Profiles of WHO and DNDi.

Folate biofortification of tomato fruit.

Folate deficiency leads to neural tube defects and other human diseases, and is a global health problem. Because plants are major folate sources for humans, we have sought to enhance plant folate levels (biofortification). Folates are synthesized from pteridine, p-aminobenzoate (PABA), and glutamate precursors. Previously, we increased pteridine production in tomato fruit up to 140-fold by overexpressing GTP cyclohydrolase I, the first enzyme of pteridine synthesis. This strategy increased folate levels 2-fold, but engineered fruit were PABA-depleted. We report here the engineering of fruit-specific overexpression of aminodeoxychorismate synthase, which catalyzes the first step of PABA synthesis. The resulting fruit contained an average of 19-fold more PABA than controls. When transgenic PABA- and pteridine-overproduction traits were combined by crossing, vine-ripened fruit accumulated up to 25-fold more folate than controls. Folate accumulation was almost as high (up to 15-fold) in fruit harvested green and ripened by ethylene-gassing, as occurs in commerce. The accumulated folates showed normal proportions of one-carbon forms, with 5-methyltetrahydrofolate the most abundant, but were less extensively polyglutamylated than controls. Folate concentrations in developing fruit did not change in controls, but increased continuously throughout ripening in transgenic fruit. Pteridine and PABA levels in transgenic fruit were >20-fold higher than in controls, but the pathway intermediates dihydropteroate and dihydrofolate did not accumulate, pointing to a flux constraint at the dihydropteroate synthesis step. The folate levels we achieved provide the complete adult daily requirement in less than one standard serving.

Regulation of pyrG expression in Bacillus subtilis: CTP-regulated antitermination and reiterative transcription with pyrG templates in vitro.

The regulation of pyrG expression in a group of low GC Gram-positive bacteria was previously shown to be mediated by a novel form of transcription attenuation in which low levels of intracellular CTP induce reiterative addition of G residues at position +4 in the 5' end of the pyrG mRNA, which is encoded as pppGGGC. . . . The poly(G) sequences formed under these conditions act to prevent attenuation by base pairing with the C- and U-rich 5' strand of a downstream terminator stem-loop located in the pyrG leader. In this work we document the reconstitution of this regulatory system in vitro using only the native pyrG DNA template, RNA polymerase and appropriate concentrations of ribonucleotides. CTP-regulated reiterative transcription producing 5'-poly(G) tracts and regulation of transcription termination at the pyrG attenuator by CTP were demonstrated. Mutations in the native pyrG template that altered reiterative transcription and attenuation in vivo resulted in alternations in expression in the in vitro transcription system that were predicted by the mechanism described above. These findings provide strong experimental support for the proposed reiterative transcription/antitermination mechanism and confirm that no trans-acting regulatory protein is required for pyrG regulation.