Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1β converting enzyme cDNA

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1β converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-γ. First, we compared the levels of IFN-γ secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-γ production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-γ levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8+ T cell-mediated and partially IFN-γ-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8+ T cells and IFN-γ-dependent pathways.

Serine racemase: A glial enzyme synthesizing d-serine to regulate glutamate-N-methyl-d-aspartate neurotransmission

Although d amino acids are prominent in bacteria, they generally are thought not to occur in mammals. Recently, high levels of d-serine have been found in mammalian brain where it activates glutamate/N-methyl-d-aspartate receptors by interacting with the “glycine site” of the receptor. Because amino acid racemases are thought to be restricted to bacteria and insects, the origin of d-serine in mammals has been puzzling. We now report cloning and expression of serine racemase, an enzyme catalyzing the formation of d-serine from l-serine. Serine racemase is a protein representing an additional family of pyridoxal-5′ phosphate-dependent enzymes in eukaryotes. The enzyme is enriched in rat brain where it occurs in glial cells that possess high levels of d-serine in vivo. Occurrence of serine racemase in the brain demonstrates the conservation of d-amino acid metabolism in mammals with implications for the regulation of N-methyl-d-aspartate neurotransmission through glia-neuronal interactions.

Structural distribution of stability in a thermophilic enzyme

Stability parameters for individual residues in Thermus thermophilus cysteine-free RNase H were determined by native state hydrogen exchange, thus providing a unique comparison of regional thermodynamics between thermophilic and mesophilic homologues. The general distribution of stability in the thermophilic protein is similar to that of its mesophilic homologue, with a proportional increase in stability for almost all residues. As a consequence, the residue-specific stabilities of the two proteins are remarkably similar under conditions where their global stabilities are the same. These results indicate that T. thermophilus RNase H is stabilized in a delocalized fashion, preserving a finely tuned balance of stabilizing interactions throughout the structure. Therefore, although protein stability can be altered by single amino acid substitution, evolution for optimal function may require more subtle and delocalized mechanisms.

A link between protein structure and enzyme catalyzed hydrogen tunneling

We present evidence that the size of an active site side chain may modulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction. Primary and secondary kH/kT and kD/kT kinetic isotope effects have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25°C. As reported in earlier studies, the relationship between secondary kH/kT and kD/kT isotope effects provides a sensitive probe for deviations from classical behavior. In the present work, catalytic efficiency and the extent of hydrogen tunneling have been correlated for the alcohol dehydrogenase-catalyzed hydride transfer among a group of site-directed mutants at position 203. Val-203 interacts with the opposite face of the cofactor NAD+ from the alcohol substrate. The reduction in size of this residue is correlated with diminished tunneling and a two orders of magnitude decrease in catalytic efficiency. Comparison of the x-ray crystal structures of a ternary complex of a high-tunneling (Phe-93 → Trp) and a low-tunneling (Val-203 → Ala) mutant provides a structural basis for the observed effects, demonstrating an increase in the hydrogen transfer distance for the low-tunneling mutant. The Val-203 → Ala ternary complex crystal structure also shows a hyperclosed interdomain geometry relative to the wild-type and the Phe-93 → Trp mutant ternary complex structures. This demonstrates a flexibility in interdomain movement that could potentially narrow the distance between the donor and acceptor carbons in the native enzyme and may enhance the role of tunneling in the hydride transfer reaction.

Mammalian capping enzyme complements mutant Saccharomyces cerevisiae lacking mRNA guanylyltransferase and selectively binds the elongating form of RNA polymerase II

5′-Capping is an early mRNA modification that has important consequences for downstream events in gene expression. We have isolated mammalian cDNAs encoding capping enzyme. They contain the sequence motifs characteristic of the nucleotidyl transferase superfamily. The predicted mouse and human enzymes consist of 597 amino acids and are 95% identical. Mouse cDNA directed synthesis of a guanylylated 68-kDa polypeptide that also contained RNA 5′-triphosphatase activity and catalyzed formation of RNA 5′-terminal GpppG. A haploid strain of Saccharomyces cerevisiae lacking mRNA guanylyltransferase was complemented for growth by the mouse cDNA. Conversion of Lys-294 in the KXDG-conserved motif eliminated both guanylylation and complementation, identifying it as the active site. The K294A mutant retained RNA 5′-triphosphatase activity, which was eliminated by N-terminal truncation. Full-length capping enzyme and an active C-terminal fragment bound to the elongating form and not to the initiating form of polymerase. The results document functional conservation of eukaryotic mRNA guanylyltransferases from yeast to mammals and indicate that the phosphorylated C-terminal domain of RNA polymerase II couples capping to transcription elongation. These results also explain the selective capping of RNA polymerase II transcripts.

A functional homolog of a yeast tRNA splicing enzyme is conserved in higher eukaryotes and in Escherichia coli

tRNA splicing in the yeast Saccharomyces cerevisiae requires an endonuclease to excise the intron, tRNA ligase to join the tRNA half-molecules, and 2′-phosphotransferase to transfer the splice junction 2′-phosphate from ligated tRNA to NAD, producing ADP ribose 1′′–2′′ cyclic phosphate (Appr>p). We show here that functional 2′-phosphotransferases are found throughout eukaryotes, occurring in two widely divergent yeasts (Candida albicans and Schizosaccharomyces pombe), a plant (Arabidopsis thaliana), and mammals (Mus musculus); this finding is consistent with a role for the enzyme, acting in concert with ligase, to splice tRNA or other RNA molecules. Surprisingly, functional 2′-phosphotransferase is found also in the bacterium Escherichia coli, which does not have any known introns of this class, and does not appear to have a ligase that generates junctions with a 2′-phosphate. Analysis of the database shows that likely members of the 2′-phosphotransferase family are found also in one other bacterium (Pseudomonas aeruginosa) and two archaeal species (Archaeoglobus fulgidus and Pyrococcus horikoshii). Phylogenetic analysis reveals no evidence for recent horizontal transfer of the 2′-phosphotransferase into Eubacteria, suggesting that the 2′-phosphotransferase has been present there since close to the time that the three kingdoms diverged. Although 2′-phosphotransferase is not present in all Eubacteria, and a gene disruption experiment demonstrates that the protein is not essential in E. coli, the continued presence of 2′-phosphotransferase in Eubacteria over large evolutionary times argues for an important role for the protein.

Overexpression of thioredoxin h leads to enhanced activity of starch debranching enzyme (pullulanase) in barley grain

Biochemically active wheat thioredoxin h has been overexpressed in the endosperm of transgenic barley grain. Two DNA constructs containing the wheat thioredoxin h gene (wtrxh) were used for transformation; each contained wtrxh fused to an endosperm-specific B1-hordein promoter either with or without a signal peptide sequence for targeting to the protein body. Twenty-two stable, independently transformed regenerable lines were obtained by selecting with the herbicide bialaphos to test for the presence of the bar herbicide resistance gene on a cotransformed plasmid; all were positive for this gene. The presence of wtrxh was confirmed in 20 lines by PCR analysis, and the identity and level of expression of wheat thioredoxin h was assessed by immunoblots. Although levels varied among the different transgenic events, wheat thioredoxin h was consistently highly expressed (up to 30-fold) in the transgenic grain. Transgenic lines transformed with the B1-hordein promoter with a signal peptide sequence produced a higher level of wheat thioredoxin h on average than those without a signal sequence. The overexpression of thioredoxin h in the endosperm of germinated grain effected up to a 4-fold increase in the activity of the starch debranching enzyme, pullulanase (limit dextrinase), the enzyme that specifically cleaves α-1,6 linkages in starch. These results raise the question of how thioredoxin h enhances the activity of pullulanase because it was found that the inhibitor had become inactive before the enzyme showed appreciable activity.

Apoprotein B100 has a prolonged interaction with the translocon during which its lipidation and translocation change from dependence on the microsomal triglyceride transfer protein to independence

When lipid synthesis is limited in HepG2 cells, apoprotein B100 (apoB100) is not secreted but rapidly degraded by the ubiquitin-proteasome pathway. To investigate apoB100 biosynthesis and secretion further, the physical and functional states of apoB100 destined for either degradation or lipoprotein assembly were studied under conditions in which lipid synthesis, proteasomal activity, and microsomal triglyceride transfer protein (MTP) lipid-transfer activity were varied. Cells were pretreated with a proteasomal inhibitor (which remained with the cells throughout the experiment) and radiolabeled for 15 min. During the chase period, labeled apoB100 remained associated with the microsomes. Furthermore, by crosslinking sec61β to apoB100, we showed that apoB100 remained close to the translocon at the same time apoB100–ubiquitin conjugates could be detected. When lipid synthesis and lipoprotein assembly/secretion were stimulated by adding oleic acid (OA) to the chase medium, apoB100 was deubiquitinated, and its interaction with sec61β was disrupted, signifying completion of translocation concomitant with the formation of lipoprotein particles. MTP participates in apoB100 translocation and lipoprotein assembly. In the presence of OA, when MTP lipid-transfer activity was inhibited at the end of pulse labeling, apoB100 secretion was abolished. In contrast, when the labeled apoB100 was allowed to accumulate in the cell for 60 min before adding OA and the inhibitor, apoB100 lipidation and secretion were no longer impaired. Overall, the data imply that during most of its association with the endoplasmic reticulum, apoB100 is close to or within the translocon and is accessible to both the ubiquitin-proteasome and lipoprotein-assembly pathways. Furthermore, MTP lipid-transfer activity seems to be necessary only for early translocation and lipidation events.

Roles for mannitol and mannitol dehydrogenase in active oxygen-mediated plant defense

Reactive oxygen species (ROS) are both signal molecules and direct participants in plant defense against pathogens. Many fungi synthesize mannitol, a potent quencher of ROS, and there is growing evidence that at least some phytopathogenic fungi use mannitol to suppress ROS-mediated plant defenses. Here we show induction of mannitol production and secretion in the phytopathogenic fungus Alternaria alternata in the presence of host-plant extracts. Conversely, we show that the catabolic enzyme mannitol dehydrogenase is induced in a non-mannitol-producing plant in response to both fungal infection and specific inducers of plant defense responses. This provides a mechanism whereby the plant can counteract fungal suppression of ROS-mediated defenses by catabolizing mannitol of fungal origin.

Crystal structure of glutamate-1-semialdehyde aminomutase: An α2-dimeric vitamin B6-dependent enzyme with asymmetry in structure and active site reactivity

The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8), an α2-dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-Å resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme’s unusual specificity and functional properties. The overall fold is that of aspartate aminotransferase and related B6 enzymes, but it also has specific features. The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32. Glu-406 is suitably positioned to repel α-carboxylic acids, thereby suggesting a basis for the enzyme’s reaction specificity. The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153–181. In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153–181 are disordered. In the other subunit in which the cofactor is not covalently bound, residues 153–181 are well defined. Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution. Analysis of absorption spectra during reduction provided evidence of communication between the subunits. The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153–181 remains.

Homospermidine synthase, the first pathway-specific enzyme of pyrrolizidine alkaloid biosynthesis, evolved from deoxyhypusine synthase

Pyrrolizidine alkaloids are preformed plant defense compounds with sporadic phylogenetic distribution. They are thought to have evolved in response to the selective pressure of herbivory. The first pathway-specific intermediate of these alkaloids is the rare polyamine homospermidine, which is synthesized by homospermidine synthase (HSS). The HSS gene from Senecio vernalis was cloned and shown to be derived from the deoxyhypusine synthase (DHS) gene, which is highly conserved among all eukaryotes and archaebacteria. DHS catalyzes the first step in the activation of translation initiation factor 5A (eIF5A), which is essential for eukaryotic cell proliferation and which acts as a cofactor of the HIV-1 Rev regulatory protein. Sequence comparison provides direct evidence for the evolutionary recruitment of an essential gene of primary metabolism (DHS) for the origin of the committing step (HSS) in the biosynthesis of pyrrolizidine alkaloids.

Carbonic anhydrase is an ancient enzyme widespread in prokaryotes

Carbonic anhydrases catalyze the reversible hydration of CO2 and are ubiquitous in highly evolved eukaryotes. The recent identification of a third class of carbonic anhydrase (γ class) in a methanoarchaeon and our present finding that the β class also extends into thermophilic species from the Archaea domain led us to initiate a systematic search for these enzymes in metabolically and phylogenetically diverse prokaryotes. Here we show that carbonic anhydrase is widespread in the Archaea and Bacteria domains, and is an ancient enzyme. The occurrence in chemolithoautotrophic species occupying deep branches of the universal phylogenetic tree suggests a role for this enzyme in the proposed autotrophic origin of life. The presence of the β and γ classes in metabolically diverse species spanning the Archaea and Bacteria domains demonstrates that carbonic anhydrases have a far more extensive and fundamental role in prokaryotic biology than previously recognized.

Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: Finding of a lipase motif and the induction by methyl jasmonate

Chlorophyllase (Chlase) is the first enzyme involved in chlorophyll (Chl) degradation and catalyzes the hydrolysis of ester bond to yield chlorophyllide and phytol. In the present study, we isolated the Chlase cDNA. We synthesized degenerate oligo DNA probes based on the internal amino acid sequences of purified Chlase from Chenopodium album, screened the C. album cDNA library, and cloned a cDNA (CaCLH, C. album chlorophyll-chlorophyllido hydrolase). The deduced amino acid sequence (347 aa residues) had a lipase motif overlapping with an ATP/GTP-binding motif (P-loop). CaCLH possibly was localized in the extraplastidic part of the cell, because a putative signal sequence for endoplasmic reticulum is at the N terminus. The amino acid sequence shared 37% identity with a function-unknown gene whose mRNA is inducible by coronatine and methyl jasmonate (MeJA) in Arabidopsis thaliana (AtCLH1). We expressed the gene products of AtCLH1 and of CaCLH in Escherichia coli, and they similarly exhibited Chlase activity. Moreover, we isolated another full-length cDNA based on an Arabidopsis genomic fragment and expressed it in E. coli, demonstrating the presence of the second Arabidopsis CLH gene (AtCLH2). No typical feature of signal sequence was identified in AtCLH1, whereas AtCLH2 had a typical signal sequence for chloroplast. AtCLH1 mRNA was induced rapidly by a treatment of MeJA, which is known to promote senescence and Chl degradation in plants, and a high mRNA level was maintained up to 9 h. AtCLH2, however, did not respond to MeJA.

Human fatty acid synthase: Assembling recombinant halves of the fatty acid synthase subunit protein reconstitutes enzyme activity

Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (Mr ≈ 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the β-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 Å of the pantetheinyl-SH of the second subunit (where the malonyl group is bound). This arrangement generates two active centers for fatty acid synthesis and predicts that if we have two appropriate halves of the monomer, we should be able to reconstitute an active fatty acid-synthesizing site. We cloned, expressed, and purified catalytically active thioredoxin (TRX) fusion proteins of the NH2-terminal half of the human FAS subunit protein (TRX-hFAS-dI; residues 1–1,297; Mr ≈ 166) and of the C-terminal half (TRX-hFAS-dII-III; residues 1,296–2,504; Mr ≈ 155). Adding equivalent amounts of TRX-hFAS-dI and TRX-hFAS-dII-III to a reaction mixture containing acetyl-CoA, malonyl-CoA, and NADPH resulted in the synthesis of long-chain fatty acids. The rate of synthesis was dependent upon the presence of both recombinant proteins and reached a constant level when they were present in equivalent amounts, indicating that the reconstitution of an active fatty acid-synthesizing site required the presence of every partial activity associated with the subunit protein. Analyses of the product acids revealed myristate to be the most abundant with small amounts of palmitate and stearate, possibly because of the way the fused recombinant proteins interacted with each other so that the thioesterase hydrolyzed the acyl group in its myristoyl state. The successful reconstitution of the human FAS activity from its domain I and domains II and III fully supports our model for the structure–function relationship of FAS in animal tissues.

Genetic interactions with Rap1 and Ras1 reveal a second function for the Fat facets deubiquitinating enzyme in Drosophila eye development

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential very early in eye development, prior to facet assembly, to limit the number of photoreceptor cells in each facet of the compound eye to eight. The Fat facets protein facilitates the production of a signal in cells outside the developing facets that inhibits neural development of particular facet precursor cells. Novel gain-of-function mutations in the Drosophila Rap1 and Ras1 genes are described herein that interact genetically with fat facets mutations. Analysis of these genetic interactions reveals that Fat facets has an additional function later in eye development involving Rap1 and Ras1 proteins. Moreover, the results suggest that undifferentiated cells outside the facet continue to influence facet assembly later in eye development.