CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery.

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

Specific enzymic methylation of an arginine in the experimental allergic encephalomyelitis protein from human myelin.

A cytoplasmic enzyme from guinea pig brain was shown to transfer methyl groups from S-adenosylmethionine to only one of 19 arginine residues in the basic protein from human brain. The products were omega-N-monomethylarginine and omega-N,N'-dimethylarginine. These methylated arginines are adjacent to the main encephalitogenic determinant in the protein. Methylation may aid in the transfer of this region of the protein into the nonpolar environment within myelin and in maintaining the integrity of myelin.

A kinetic analysis of substrate recognition by uracil-DNA glycosylase from herpes simplex virus type 1.

Uracil-DNA glycosylase (UDG) is responsible for the removal of uracil from DNA. It has previously been demonstrated that UDG exhibits some sequence dependence in its activity, although this has not been well characterised. This study has investigated the sequence-dependent activity of UDG from herpes simplex virus type-1 (HSV-1). A more detailed analysis has been possible by using both kinetic and binding assays with a variety of different oligonucleotide substrates. The target uracil has been placed in substrates with either A-T-rich or G-C-rich flanking sequences and analyses have been performed on both the single- and double-stranded forms of each substrate. In the latter the uracil has been placed in both a U.A base pair and a U.G mismatch. It is observed that the sequences flanking the target uracil have a greater effect on UDG activity than the partner base of the uracil. Furthermore, the sequence context effects extend to single-stranded DNA. Systematic examination of the kinetics and binding of UDG with these different substrates has enabled us to examine the origin of the sequence preferences. We conclude that the damage recognition step in the HSV-1 UDG reaction pathway is modulated by local DNA sequence.

Structural analysis of a mutational hot-spot in the EcoRV restriction endonuclease: a catalytic role for a main chain carbonyl group.

Following random mutagenesis of the Eco RV endonuclease, a high proportion of the null mutants carry substitutions at Gln69. Such mutants display reduced rates for the DNA cleavage step in the reaction pathway, yet the crystal structures of wild-type Eco RV fail to explain why Gln69 is crucial for activity. In this study, crystal structures were determined for two mutants of Eco RV, with Leu or Glu at residue 69, bound to specific DNA. The structures of the mutants are similar to the native protein and no function can be ascribed to the side chain of the amino acid at this locus. Instead, the structures of the mutant proteins suggest that the catalytic defect is due to the positioning of the main chain carbonyl group. In the enzyme-substrate complex for Eco RV, the main chain carbonyl of Gln69 makes no interactions with catalytic functions but, in the enzyme-product complex, it coordinates a metal ion bound to the newly liberated 5'-phosphate. This re-positioning may be hindered in the mutant proteins. Molecular dynamics calculations indicate that the metal on the phosphoryl oxygen interacts with the carbonyl group upon forming the pentavalent intermediate during phosphodiester hydrolysis. A main chain carbonyl may thus play a role in catalysis by Eco RV.

Binding of gastrin(17) to human gastric carcinoma cell lines.

The hormone gastrin stimulates acid secretion by gastric parietal cells and acts as a growth factor for the gastric mucosa. Gastrin receptors with dissociation constants of approximately 0.5 nM have been detected on isolated gastric parietal cells, and on some cell lines derived from colon carcinomas. We now report that gastrin is also bound by five cell lines derived from human gastric carcinomas, but that the affinities of these lines for gastrin range from 0.2 to 1.3 microM. Cholecystokinin8 binds to the cell line Okajima with an affinity similar to gastrin17, while shorter gastrin analogues bind with reduced affinity. Binding of gastrin is unaffected by acetylcholine, histamine, or a number of other hormones with the exception of insulin which inhibits binding with an IC50 value of 0.5 microM. The ability to bind gastrin with affinities in the microM range appears to be a property widespread among other tumor cell lines.

Measurement of gastrin and transforming growth factor alpha messenger RNA levels in colonic carcinoma cell lines by quantitative polymerase chain reaction.

A synthetic DNA template has been constructed that is suitable for the quantitation of mRNAs encoding gastrin, transforming growth factor alpha (TGF alpha), cholecystokinin, and the 78-kDa gastrin-binding protein. The template was used to measure levels of gastrin and TGF alpha mRNA in 7 colonic and 2 gastric carcinoma cell lines by the polymerase chain reaction. All lines produced detectable gastrin and TGF alpha mRNA with amounts varying between 2.1 and 540 molecules of gastrin mRNA/10(3) cells and 1.1 and 28 molecules of TGF alpha mRNA/10(3) cells. These results are consistent with the hypothesis that both gastrin and TGF alpha act as autocrine growth factors in colon carcinoma cell lines.

Kinetic studies on the mechanism of chorismate mutase/prephenate dehydratase from Escherichia coli K12.

The effect of pH on chorismate mutase/prephenate dehydratase (chorismate pyruvate mutase/prephenate hydro-lyase (decarboxylating) EC 5.4.99.5/EC 4.2.1.51) from Escherichia coli K12 has been studied. While the maximum velocity of both activities is independent of pH, Km for chorismate or prephenate shows a complex pH dependence. Differences in mutase activity in acetate/phosphate/borate and citrate/phosphate/borate buffers were traced to inhibition by citrate. When a variety of analogues of citrate were tested as possible inhibitors of the enzyme, several were found to inhibit mutase and dehydratase activities to different extents, and by different mechanisms. Thus citrate competitively inhibits mutase activity, but inhibits dehydratase activity by either a non-competitive or an uncompetitive mechanism. Conversely, cis- and trans-aconitate competitively inhibit dehydratase activity, but are partially competitive inhibitors of mutase activity. The differential effects of these inhibitors on the two activities are consistent with the existence of two distinct active sites, but additionally suggest some degree of interconnection between them. The implications of these results for possible mechanisms of catalysis by chorismate mutase/prephenate dehydratase are discussed.

Structures of the human cDNA and gene encoding the 78 kDa gastrin-binding protein and of a related pseudogene.

The nucleotide sequence encoding the human 78 kDa gastrin binding protein (GBP) has been deduced from overlapping fragments generated by the polymerase chain reaction with oligonucleotides based on the sequence of the porcine GBP (Mantamadiotis, T. et al. (1993) Biochim. Biophys. Acta 1170, 211-215) and cDNA from the colonic carcinoma cell line LIM 1215 as template. The mature human GBP is 90% identical to the porcine GBP. Clones encoding the human GBP gene, which contains 19 exons, have been isolated from human genomic libraries. The positions of the exon/intron junctions are completely different from the junctions in the gene encoding the related peroxisomal trifunctional enzyme. Clones encoding a related pseudogene have also been isolated and sequenced.

Structural requirements for the binding of non-steroidal anti-inflammatory drugs to the 78 kDa gastrin binding protein.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit the proliferation of colorectal carcinoma cell lines in vitro and reduce the risk of colorectal carcinoma in vivo. The good correlation observed between the potency of NSAIDs as inhibitors of colorectal carcinoma cell proliferation and as antagonists of a 78 kDa gastrin binding protein (GBP) suggested that blockade of the GBP might contribute to the anti-proliferative effects of NSAIDs [G.S. Baldwin, V.J. Murphy, Z. Yang, T. Hashimoto, J. Pharmacol. Exp. Ther. 286 (1998) 1110-1114]. The most potent NSAID investigated was sulindac sulphide, which had an IC50 value of 40 microM. In order to investigate the structural requirements for binding to the GBP, 26 analogues of sulindac sulphide and sulindac sulphoxide were tested for their ability to inhibit the binding of iodinated gastrin to the GBP. Six of the analogues inhibited gastrin binding by more than 50% at a concentration of 1 mM. The IC50 values estimated by computer fitting of titration data were in the range of 280-940 microM. Comparison of the analogue structures suggests that a substituent with a carboxyl group is preferred in the R2 position. In addition the location of the NSAID binding site within the GBP structure was investigated. NSAIDs bound to both the N- and C-terminal halves of the GBP, and the affinities determined were similar to the values previously reported for the full-length GBP. The results reported herein represent the first step in the rational design of more potent GBP antagonists, some of which may be useful for the treatment of colorectal carcinoma.

Amino acid sequences of soluble tryptic peptides of chorismate mutase/prephenate dehydratase from Escherichia coli K12.

The amino acid sequences of 28 soluble tryptic peptides from chorismate mutase/prephenate dehydratase from Escherichia coli K12 have been determined. Together with the four unique cysteine-containing peptides sequenced by Gething and Davidson ((1976) Eur. J. Biochem. 71, 327-336) this accounts for approximately 75% of the total sequence expected for this protein. A high frequency of identify between some of the peptides suggests the possibility of gene duplication during the evolution of the structural gene for the enzyme.

Nucleotide sequence encoding a novel member of the hydratase/dehydrogenase family.

The nucleotide sequence encoding a novel member of the family of fatty acid oxidation enzymes has been determined. Clones were generated from porcine gastric corpus cDNA by application of the polymerase chain reaction (PCR) using oligodeoxyribonucleotides based on the amino acid sequence of a 78 kDa gastrin-binding protein isolated from porcine gastric mucosal membranes (Baldwin et al., J. Biol. Chem. 261 (1986) 12252-12257). Clones encoding the 3' and 5' ends of the cDNA were then isolated by conventional screening of a porcine liver cDNA library, and by application of anchored PCR, respectively. The composite cDNA of 2744 nucleotides encoded a protein of 763 amino acids, the sequence of which was related to a rat peroxisomal trifunctional enzyme, delta 3, delta 2-enoyl-CoA isomerase/enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, and to other members of the same enzyme family. Northern blots indicated that the 3-kb mRNA encoding the novel protein was abundant in gastric corpus and liver, with lower amounts also present in gastric antrum and forestomach.

Reduction in platelet-derived growth factor receptor mRNA in v-src-transformed fibroblasts.

The status of the platelet-derived growth factor (PDGF) receptor in normal rat kidney (NRK) fibroblasts and in NRK fibroblasts transformed by the v-src oncogene or the polyoma middle T (pmt) antigen has been compared. v-src-NRK cells have 7-fold fewer surface binding sites for PDGF than NRK cells, but the affinity of the residual receptors for PDGF is reduced only 2-fold. Levels of the PDGF receptor measured by Western blotting or in an autophosphorylation assay in vitro are 8- and 4-fold lower respectively in v-src-NRK cells than in NRK cells. No PDGF-induced phosphorylation of the PDGF receptor is apparent after 32P-labelling of intact v-src-NRK cells, implying that the reduction in PDGF receptor levels is not a consequence of production of autocrine PDGF. A 10-fold reduction in the amount of mRNA for the PDGF receptor is also observed in v-src-NRK cells. No decrease in PDGF receptor protein or mRNA levels is observed in pmt-NRK cells. We conclude that levels of the PDGF receptor in v-src-transformed NRK fibroblasts are modulated by reduction in the level of PDGF receptor mRNA.

Binding of pepsinogen to the 78 kDa gastrin binding protein.

An endogenous ligand of the 78 kDa gastrin-binding protein (GBP) has been purified from detergent extracts of porcine gastric mucosal membranes by ion exchange chromatography and preparative gel electrophoresis. The ligand bound to the GBP with high affinity (mean IC50 value of 0.31+/-0.09 microgram/ml, or 8 nM), as assessed by inhibition of cross-linking of iodinated gastrin2,17 to the GBP. Both the N- and C-terminal halves of the GBP, which had been expressed individually as glutathione-S-transferase fusion proteins in Escherichia coli, and purified on glutathione-agarose beads, bound the ligand. Two peptides derived from the ligand were purified by reversed-phase high-performance liquid chromatography (HPLC), and characterised by mass spectrometry and Edman sequencing. The peptides were 97% and 100% identical, respectively, to amino acids 119-157 and 199-219 of porcine pepsinogen A. Commercial samples of pepsinogen also bound to the GBP, with a mean IC50 value of 3.9+/-1. 2 micrograms/ml (100 nM). We conclude that the ligand is closely related, but not identical, to pepsinogen A.

Controlled overexpression of selected domains of the P85 subunit of phosphatidylinositol 3-kinase reverts v-Ha-Ras transformation.

Selected domains of the regulatory p85 subunit of phosphatidylinositol 3-kinase have been expressed under the control of the tetracycline transactivator in NIH 3T3 fibroblasts transformed by the v-Ha-Ras oncogene. The domains expressed were the SH3 domain, the BCR homology domain, the region between the two SH2 domains which contains the p110 binding site (the inter SH2 (IS) domain), and the C-terminal (CT) domain (containing both SH2 domains and the IS domain). The levels of IS or SH3 domain expressed in the presence of tetracycline were sufficient to reverse the transforming effects of v-Ha-Ras, and no further inhibition of proliferation was observed when expression was increased 7-fold by removal of tetracycline. In contrast inhibition of proliferation by the CT domain was observed only when the level of expression was increased 5-fold by removal of tetracycline. Overexpression of the BCR domain of p85 had no effect on v-Ha-Ras transformation. Expression of the IS domain disrupted the interaction of the p85 regulatory subunit with the p110 catalytic subunit. These results indicate that the association of the p85 subunit of PI 3-kinase with the p110 subunit is necessary for v-Ha-Ras-induced transformation in NIH 3T3 cells.

DNA cleavage by the EcoRV restriction endonuclease: pH dependence and proton transfers in catalysis.

To characterise the pH dependence of phosphodiester hydrolysis by the EcoRV endonuclease in the presence of Mn2+, single turnover reactions on a 12 bp DNA substrate were examined by stopped-flow and quench-flow methods between pH 6.0 and 8.5. At each pH value, the apparent rate constants for phosphodiester hydrolysis increased hyperbolically with the concentration of MnCl2, thus allowing values to be determined for the intrinsic rate constant at saturation with Mn2+ and the equilibrium dissociation constant for Mn2+. The equilibrium constants showed no systematic variation across the pH range tested, while the rate constants increased steeply with increasing pH up to an asymptote above pH 7.5. At low pH conditions, the gradient of a plot of log (rate constant) against pH approached a value of 2. DNA cleavage by EcoRV thus requires the de-protonation of two acidic groups. To determine whether aspartate 36 is one of the groups, mutants of EcoRV were made with other amino acid residues at position 36. Glutamate caused a partial loss of activity, while all other replacements gave near-zero activities. In contrast to wild-type EcoRV, the mutant with glutamate required the de-protonation of only one acidic group for DNA cleavage. A mechanism for EcoRV is proposed in which the water molecule that hydrolyses the phosphodiester bond is de-protonated by two Bronsted bases, probably the ionised forms of aspartate 36 and glutamate 45.

DNA cleavage by the EcoRV restriction endonuclease: roles of divalent metal ions in specificity and catalysis.

The roles of divalent metal ions in DNA cleavage by the EcoRV endonuclease were studied by using Co2+ or Mn2+ as substitutes for the natural cofactor Mg2+. In steady-state experiments with a 12 bp oligonucleotide substrate, Co2+ yielded a similar turnover rate to that with Mg2+, but Mn2+ gave a slower rate. Single turnovers of EcoRV on this substrate were analysed by stopped-flow and quench-flow methods, to determine the rates for the formation of the ternary enzyme-DNA-metal complex, the hydrolysis of the phosphodiester bonds and the dissociation of the cleaved DNA. With Co2+, all three steps had similar rates to those with Mg2+. In contrast, Mn2+ gave a faster rate for phosphodiester hydrolysis than either Mg2+ or Co2+, but a slower rate for product dissociation, thus accounting for its low turnover rate. Single turnovers on plasmids also yielded faster rates for substrate hydrolysis with Mn2+ compared to Mg2+ and Co2+. Since Mn2+ gave the most rapid rates for the hydrolytic step, despite being less electronegative than Co2+, the function of the metal ion at the active site of EcoRV cannot be just the polarisation of the scissile phosphate. Moreover, the minimal scheme for the Co2+-catalysed reaction requires two metal ions for DNA cleavage. The metal ions seem to be involved in the precise positioning of both the substrate and the water that acts as the attacking nucleophile and in activating that water molecule. A model is presented to account for how two metal ions might fulfil these functions.

Mechanism-based inhibition of C5-cytosine DNA methyltransferases by 2-H pyrimidinone.

DNA duplexes in which the target cytosine base is replaced by 2-H pyrimidinone have previously been shown to bind with a significantly greater affinity to C5-cytosine DNA methyltransferases than unmodified DNA. Here, it is shown that 2-H pyrimidinone, when incorporated into DNA duplexes containing the recognition sites for M.HgaI-2 and M.MspI, elicits the formation of inhibitory covalent nucleoprotein complexes. We have found that although covalent complexes are formed between 2-H pyrimidinone-modified DNA and both M.HgaI-2 and M.MspI, the kinetics of complex formation are quite distinct in each case. Moreover, the formation of a covalent complex is still observed between 2-H pyrimidinone DNA and M.MspI in which the active-site cysteine residue is replaced by serine or threonine. Covalent complex formation between M.MspI and 2-H pyrimidinone occurs as a direct result of nucleophilic attack by the residue at the catalytic position, which is enhanced by the absence of the 4-amino function in the base. The substitution of the catalytic cysteine residue by tyrosine or chemical modification of the wild-type enzyme with N-ethylmaleimide, abolishes covalent interaction. Nevertheless the 2-H pyrimidinone-substituted duplex still binds to M.MspI with a greater affinity than a standard cognate duplex, since the 2-H pyrimidinone base is mis-paired with guanine.

Ligand-induced conformational states of the cytosine-specific DNA methyltransferase M.HgaI-2.

The interaction of one of the two DNA methyltransferases encoded by the HgaI restriction and modification system, M.HgaI-2, with substrates and substrate analogues is described. Circular dichroism spectroscopy has been used to demonstrate that addition of the methyl donor, S-adenosyl-L-methionine and the inhibitory substrate analogue sinefungin, both induce conformational transitions in the protein in the absence of DNA. Moreover, the addition of DNA is shown to enhance the apparent secondary structure of M.HgaI-2 whilst addition of sinefungin or S-adenosyl-L-methionine reduces apparent secondary structure. The circular dichroism spectrum of the abortive complex between the enzyme, DNA and sinefungin is dominated by the conformational properties of the binary complex of enzyme and sinefungin alone. Addition of a specific oligodeoxynucleotide duplex in which the target cytosine is replaced by a pyrimidinone, leads to a further ligand induced conformational transition as determined by electrophoretic analysis. The addition of sinefungin, or S-adenosyl-L-methionine, to M.HgaI-2 bound to the reactive oligodeoxynucleotide duplex, leads to yet another conformational transition in the protein as determined by the differential susceptibility of ternary and binary complexes to proteolysis. These experiments identify at least six ligand-inducible conformational states of M.HgaI-2 and, in view of the sequence similarity amongst this class of enzymes, suggest that conformational flexibility is a general feature of C-5 cytosine-specific DNA methyltransferases. Moreover, the substitution of the target cytosine by a pyrimidinone mimics the effect of 5-azacytosine incorporation into DNA.

Gastrin and gastrin receptor antagonists bind to both N- and C-terminal halves of the 78 kDa gastrin-binding protein.

A 78 kDa gastrin-binding protein (GBP) has previously been identified as the target of the anti-proliferative effects of non-selective gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines. The GBP was related in sequence to a family of fatty acid oxidation enzymes possessing enoyl CoA hydratase and 3-hydroxyacyl CoA dehydrogenase activity. This study aims to define the binding site for gastrin and gastrin antagonists in greater detail. The N- and C-terminal halves of the porcine GBP were expressed independently as glutathione S-transferase fusion proteins in E. coli. Affinities of gastrin and gastrin antagonists for the fusion proteins were measured by competition for 125I-[Nle15]-gastrin binding in a covalent cross-linking assay. The N- and C-terminal fusion proteins bound gastrin with affinities of 9.9 +/- 6.1 and 71 +/- 48 microM, respectively (n = 3). These values were 40-fold and 300-fold lower than the affinity of the full-length GBP for gastrin (0.23 +/- 0.15 microM). In contrast, the affinities of the N- and C-terminal halves for the antagonists proglumide (22 +/- 13 and 10 +/- 4 mM, respectively) and benzotript (350 +/- 90 and 400 +/- 160 micro M, respectively) were similar to each other and to the affinities of proglumide and benzotript for the full-length GBP (5.1 +/- 3.6 mM and 200 +/- 120 microM, respectively). It is concluded that proglumide and benzotript bind independently to both the hydratase and dehydrogenase active sites of the GBP, while a single molecule of gastrin may bind simultaneously to both active sites. A model is proposed which is consistent with these data, and which will assist in the development of more potent and selective GBP antagonists.

Overexpression of sense or antisense human gastrin mRNA does not affect proliferation of normal rat kidney fibroblasts.

Progastrin-derived peptides have been reported to stimulate mitogenesis in Swiss 3T3 fibroblasts [P. Singh, A. Owlia, R. Espeijo, B. Dai, Novel gastrin receptors mediate mitogenic effects of gastrin and processing intermediates of gastrin on Swiss 3T3 fibroblasts: Absence of detectable cholecystokinin (CCK)-A and CCK-B receptors. J. Biol. Chem. 270 (1995) 8429-8438]. The aim of the present study was to determine the generality of these findings, by investigating the effect of endogenous and exogenous progastrin-derived peptides on the proliferation of the normal rat kidney fibroblast cell line NRK. Levels of endogenous progastrin-derived peptides were modified by stable transfection of NRK cells with tetracycline-repressible plasmids containing sequences encoding human gastrin in either the sense or antisense orientation. Expression of sense and antisense gastrin mRNA was demonstrated by reverse transcriptase PCR and by radioimmunoassay, and cell proliferation rates were determined by the colorimetric MTT assay. Sense clones produced full length human progastrin, but significant quantities of glycine-extended or amidated gastrin17 were not detected. Concentrations of endogenous rat progastrin in antisense clones were significantly lower than concentrations in clones transfected with vector only. However no difference in proliferation rate was observed between sense, antisense and vector-transfected clones. No stimulation of proliferation was observed in synchronised untransfected NRK cells after supplementation of media with gastrin17 or gastrin17gly in the concentration range 0.3 to 100 nM. Our results do not provide evidence in support of the hypothesis that endogenous or exogenous progastrin-derived peptides act as growth factors in NRK fibroblasts.