Products of Proline Catabolism Can Induce Osmotically Regulated Genes in Rice1

Many plants accumulate high levels of free proline (Pro) in response to osmotic stress. This imino acid is widely believed to function as a protector or stabilizer of enzymes or membrane structures that are sensitive to dehydration or ionically induced damage. The present study provides evidence that the synthesis of Pro may have an additional effect. We found that intermediates in Pro biosynthesis and catabolism such as glutamine and Δ1-pyrroline-5-carboxylic acid (P5C) can increase the expression of several osmotically regulated genes in rice (Oryza sativa L.), including salT and dhn4. One millimolar P5C or its analog, 3,4-dehydroproline, produced a greater effect on gene expression than 1 mm l-Pro or 75 mm NaCl. These chemicals did not induce hsp70, S-adenosylmethionine synthetase, or another osmotically induced gene, Em, to any significant extent. Unlike NaCl, gene induction by P5C did not depend on the normal levels of either de novo protein synthesis or respiration, and did not raise abscisic acid levels significantly. P5C- and 3,4-dehydroproline-treated plants consumed less O2, had reduced NADPH levels, had increased NADH levels, and accumulated many osmolytes associated with osmotically stressed rice. These experiments indicate that osmotically induced increases in the concentrations of one or more intermediates in Pro metabolism could be influencing some of the characteristic responses to osmotic stress.

Fatty Acid Elongation Is Independent of Acyl-Coenzyme A Synthetase Activities in Leek and Brassica napus1

In both animal and plant acyl elongation systems, it has been proposed that fatty acids are first activated to acyl-coenzyme A (CoA) before their elongation, and that the ATP dependence of fatty acid elongation is evidence of acyl-CoA synthetase involvement. However, because CoA is not supplied in standard fatty acid elongation assays, it is not clear if CoA-dependent acyl-CoA synthetase activity can provide levels of acyl-CoAs necessary to support typical rates of fatty acid elongation. Therefore, we examined the role of acyl-CoA synthetase in providing the primer for acyl elongation in leek (Allium porrum L.) epidermal microsomes and Brassica napus L. cv Reston oil bodies. As presented here, fatty acid elongation was independent of CoA and proceeded at maximum rates with CoA-free preparations of malonyl-CoA. We also showed that stearic acid ([1-14C]18:0)-CoA was synthesized from [1-14C]18:0 in the presence of CoA-free malonyl-CoA or acetyl-CoA, and that [1-14C]18:0-CoA synthesis under these conditions was ATP dependent. Furthermore, the appearance of [1-14C]18:0 in the acyl-CoA fraction was simultaneous with its appearance in phosphatidylcholine. These data, together with the s of a previous study (A. Hlousek-Radojcic, H. Imai, J.G. Jaworski [1995] Plant J 8: 803–809) showing that exogenous [14C]acyl-CoAs are diluted by a relatively large endogenous pool before they are elongated, strongly indicated that acyl-CoA synthetase did not play a direct role in fatty acid elongation, and that phosphatidylcholine or another glycerolipid was a more likely source of elongation primers than acyl-CoAs.

Expression cloning of Forssman glycolipid synthetase: a novel member of the histo-blood group ABO gene family.

A phenotypic cloning approach was used to isolate a canine cDNA encoding Forssman glycolipid synthetase (FS; UDP-GalNAc:globoside alpha-1,3-N-acetylgalactosaminyltransferase; EC 2.4.1.88). The deduced amino acid sequence of FS demonstrates extensive identity to three previously cloned glycosyltransferases, including the enzymes responsible for synthesis of histo-blood group A and B antigens. These three enzymes, like FS, catalyze the addition of either N-acetylgalactosamine (GalNAc) or galactose (Gal) in alpha-1,3-linkage to their respective substrates. Despite the high degree of sequence similarity among the transferases, we demonstrate that the FS cDNA encodes an enzyme capable of synthesizing Forssman glycolipid, and demonstrates no GalNAc or Gal transferase activity when closely related substrates are examined. Thus, the FS cDNA is a novel member of the histo-blood group ABO gene family that encodes glycosyltransferases with related but distinct substrate specificity. Cloning of the FS cDNA will allow a detailed dissection of the roles Forssman glycolipid plays in cellular differentiation, development, and malignant transformation.

The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase.

(+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis. The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution. It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme. This work provides the first crystal structure of a herbicide-target complex reported to date.

DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite.

The free radicals nitric oxide and superoxide anion react to form peroxynitrite (ONOO-), a highly toxic oxidant species. In vivo formation of ONOO- has been demonstrated in shock and inflammation. Herein we provide evidence that cytotoxicity in cells exposed to ONOO- is mediated by DNA strand breakage and the subsequent activation of the DNA repair enzyme poly(ADP ribose) synthetase (PARS). Exposure to ONOO- (100 microM to 1 mM) inhibited mitochondrial respiration in cultured J774 macrophages and in rat aortic smooth muscle cells. The loss of cellular respiration was rapid, peaking 1-3 h after ONOO- exposure, and reversible, with recovery after a period of 6-24 h. The inhibition of mitochondrial respiration was paralleled by a dose-dependent increase in DNA strand breakage, reaching its maximum at 20-30 min after exposure to ONOO-. We observed a dose-dependent increase in the activity of PARS in cells exposed to ONOO-. Inhibitors of PARS such as 3-aminobenzamide (1 mM) prevented the inhibition of cellular respiration in cells exposed to ONOO-. Activation of PARS by ONOO--mediated DNA strand breakage resulted in a significant decrease in intracellular energy stores, as reflected by a decline of intracellular NAD+ and ATP content. 3-Aminobenzamide prevented the loss of NAD+ and ATP in cells exposed to ONOO-. In contrast, impairment of cellular respiration by the addition of the nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine or diethyltriamine nitric oxide complex, was not associated with the development of DNA strand breaks, in concentrations up to 1 mM, and was largely refractory to PARS inhibition. Our results suggest that DNA damage and activation of PARS, an energy-consuming futile repair cycle, play a central role in ONOO--mediated cellular injury.

An alternatively spliced form of the transcription factor Sp1 containing only a single glutamine-rich transactivation domain.

Protein-protein interactions involving specific transactivation domains play a central role in gene transcription and its regulation. The promoter-specific transcription factor Sp1 contains two glutamine-rich transcriptional activation domains (A and B) that mediate direct interactions with the transcription factor TFIID complex associated with RNA polymerase II and synergistic effects involving multiple Sp1 molecules. In the present study, we report the complementary DNA sequence for an alternatively spliced form of mouse Sp1 (mSp1-S) that lacks one of the two glutamine-rich activation regions present in the full-length protein. Corresponding transcripts were identified in mouse tissues and cell lines, and an Sp1-related protein identical in size to that predicted for mSp1-S was detected in mouse nuclear extracts. Cotransfection analysis revealed that mSp1-S lacks appreciable activity at promoters containing a single Sp1 response element but is active when multiple Sp1 sites are present, suggesting synergistic interactions between multiple mSp1-S molecules. The absence of a single glutamine-rich domain does not fully explain the properties of the smaller protein and indicates that additional structural features account for its unique transcriptional activity. The functional implications of this alternatively spliced form of Sp1 are discussed.

Selenophosphate synthetase: detection in extracts of rat tissues by immunoblot assay and partial purification of the enzyme from the archaean Methanococcus vannielii.

In Escherichia coli and Salmonella typhimurium it has been shown that selenophosphate serves as the selenium donor for the conversion of seryl-tRNA to selenocysteyl-tRNA and for the synthesis of 2-selenouridine, a modified nucleoside present in tRNAs. Although selenocysteyl-tRNA also is formed in eukaryotes and is used for the specific insertion of selenocysteine into proteins, the precise mechanism of its biosynthesis from seryl-tRNA in these systems is not known. Because selenophosphate is extremely oxygen labile and difficult to identify in biological systems, we used an immunological approach to detect the possible presence of selenophosphate synthetase in mammalian tissues. With antibodies elicited to E. coli selenophosphate synthetase the enzyme was detected in extracts of rat brain, liver, kidney, and lung by immunoblotting. Especially high levels were detected in Methanococcus vannielii, a member of the domain Archaea, and the enzyme was partially purified from this source. It seems likely that the use of selenophosphate as a selenium donor is widespread in biological systems.

Incorporation of glutamine repeats makes protein oligomerize: implications for neurodegenerative diseases.

Many transcription factors and some other proteins contain glutamine repeats; their abnormal expansion has been linked to several dominantly inherited neuro-degenerative diseases. Having found that poly(L-glutamine) alone forms beta-strands held together by hydrogen bonds between their amide groups, we surmised that glutamine repeats may form polar zippers, an unusual motif for protein-protein interactions. To test this hypothesis, we have engineered a Gly-Gln10-Gly peptide into the inhibitory loop of truncated chymotrypsin inhibitor 2 (CI2), a small protein from barley seeds, by both insertion and replacement. Gel filtration resolved both mutant inhibitors into at least three fractions, which analytical ultracentrifugation identified as monomers, dimers, and trimers of the recombinant protein; the truncated wild-type CI2 formed only monomers. CD difference spectra of the dimers and trimers versus wild type indicated that their glutamine repeats formed beta-pleated sheets, while those of the monomers versus wild type were more suggestive of type I beta-turns. The CD spectra of all three fractions remained unchanged even after incubation at 70 degrees C; neither the dimers nor the trimers dissociated at this temperature. We argue that the stability of all three fractions is due to the multiplicity of hydrogen bonds between extended strands of glutamine repeats in the oligomers or within a beta-hairpin formed by the single glutamine repeat of each monomer. Pathological effects may arise when expanded glutamine repeats cause proteins to acquire excessively high affinities for each other or for other proteins with glutamine repeats.

Wide cross-species aminoacyl-tRNA synthetase replacement in vivo: yeast cytoplasmic alanine enzyme replaced by human polymyositis serum antigen.

Because of variations in tRNA sequences in evolution, tRNA synthetases either do not acylate their cognate tRNAs from other organisms or execute misacylations which can be deleterious in vivo. We report here the cloning and primary sequence of a 958-aa Saccharomyces cerevisiae alanyl-tRNA synthetase. The enzyme is a close homologue of the human and Escherichia coli enzymes, particularly in the region of the primary structure needed for aminoacylation of RNA duplex substrates based on alanine tRNA acceptor stems with a G3.U70 base pair. An ala1 disrupted allele demonstrated that the gene is essential and that, therefore, ALA1 encodes an enzyme required for cytoplasmic protein synthesis. Growth of cells harboring the ala1 disrupted allele was restored by a cDNA clone encoding human alanyl-tRNA synthetase, which is a serum antigen for many polymyositis-afflicted individuals. The human enzyme in extracts from rescued yeast was detected with autoimmune antibodies from a polymyositis patient. We conclude that, in spite of substantial differences between human and yeast tRNA sequences in evolution, strong conservation of the G3.U70 system of recognition is sufficient to yield accurate aminoacylation in vivo across wide species distances.

Isolation of a cDNA encoding human holocarboxylase synthetase by functional complementation of a biotin auxotroph of Escherichia coli.

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of the four biotin-dependent carboxylases in human cells. Patients with HCS deficiency lack activity of all four carboxylases, indicating that a single HCS is targeted to the mitochondria and cytoplasm. We isolated 21 human HCS cDNA clones, in four size classes of 2.0-4.0 kb, by complementation of an Escherichia coli birA mutant defective in biotin ligase. Expression of the cDNA clones promoted biotinylation of the bacterial biotinyl carboxyl carrier protein as well as a carboxyl-terminal fragment of the alpha subunit of human propionyl-CoA carboxylase expressed from a plasmid. The open reading frame encodes a predicted protein of 726 aa and M(r) 80,759. Northern blot analysis revealed the presence of a 5.8-kb major species and 4.0-, 4.5-, and 8.5-kb minor species of poly(A)+ RNA in human tissues. Human HCS shows specific regions of homology with the BirA protein of E. coli and the presumptive biotin ligase of Paracoccus denitrificans. Several forms of HCS mRNA are generated by alternative splicing, and as a result, two mRNA molecules bear different putative translation initiation sites. A sequence upstream of the first translation initiation site encodes a peptide structurally similar to mitochondrial presequences, but it lacks an in-frame ATG codon to direct its translation. We anticipate that alternative splicing most likely mediates the mitochondrial versus cytoplasmic expression, although the elements required for directing the enzyme to the mitochondria remain to be confirmed.

Root of the universal tree of life based on ancient aminoacyl-tRNA synthetase gene duplications.

Universal trees based on sequences of single gene homologs cannot be rooted. Iwabe et al. [Iwabe, N., Kuma, K.-I., Hasegawa, M., Osawa, S. & Miyata, T. (1989) Proc. Natl. Acad. Sci. USA 86, 9355-9359] circumvented this problem by using ancient gene duplications that predated the last common ancestor of all living things. Their separate, reciprocally rooted gene trees for elongation factors and ATPase subunits showed Bacteria (eubacteria) as branching first from the universal tree with Archaea (archaebacteria) and Eucarya (eukaryotes) as sister groups. Given its topical importance to evolutionary biology and concerns about the appropriateness of the ATPase data set, an evaluation of the universal tree root using other ancient gene duplications is essential. In this study, we derive a rooting for the universal tree using aminoacyl-tRNA synthetase genes, an extensive multigene family whose divergence likely preceded that of prokaryotes and eukaryotes. An approximately 1600-bp conserved region was sequenced from the isoleucyl-tRNA synthetases of several species representing deep evolutionary branches of eukaryotes (Nosema locustae), Bacteria (Aquifex pyrophilus and Thermotoga maritima) and Archaea (Pyrococcus furiosus and Sulfolobus acidocaldarius). In addition, a new valyl-tRNA synthetase was characterized from the protist Trichomonas vaginalis. Different phylogenetic methods were used to generate trees of isoleucyl-tRNA synthetases rooted by valyl- and leucyl-tRNA synthetases. All isoleucyl-tRNA synthetase trees showed Archaea and Eucarya as sister groups, providing strong confirmation for the universal tree rooting reported by Iwabe et al. As well, there was strong support for the monophyly (sensu Hennig) of Archaea. The valyl-tRNA synthetase gene from Tr. vaginalis clustered with other eukaryotic ValRS genes, which may have been transferred from the mitochondrial genome to the nuclear genome, suggesting that this amitochondrial trichomonad once harbored an endosymbiotic bacterium.

A novel glutamine–RNA interaction identified by screening libraries in mammalian cells

The arginine-rich motif provides a versatile framework for RNA recognition in which few amino acids other than arginine are needed to mediate specific binding. Using a mammalian screening system based on transcriptional activation by HIV Tat, we identified novel arginine-rich peptides from combinatorial libraries that bind tightly to the Rev response element of HIV. Remarkably, a single glutamine, but not asparagine, within a stretch of polyarginine can mediate high-affinity binding. These results, together with the structure of a Rev peptide-Rev response element complex, suggest that the carboxamide groups of glutamine or asparagine are well-suited to hydrogen bond to G-A base pairs and begin to establish an RNA recognition code for the arginine-rich motif. The screening approach may provide a relatively general method for screening expression libraries in mammalian cells.

Activation of microhelix charging by localized helix destabilization

We report that aminoacylation of minimal RNA helical substrates is enhanced by mismatched or unpaired nucleotides at the first position in the helix. Previously, we demonstrated that the class I methionyl-tRNA synthetase aminoacylates RNA microhelices based on the acceptor stem of initiator and elongator tRNAs with greatly reduced efficiency relative to full-length tRNA substrates. The cocrystal structure of the class I glutaminyl-tRNA synthetase with tRNAGln revealed an uncoupling of the first (1⋅72) base pair of tRNAGln, and tRNAMet was proposed by others to have a similar base-pair uncoupling when bound to methionyl-tRNA synthetase. Because the anticodon is important for efficient charging of methionine tRNA, we thought that 1⋅72 distortion is probably effected by the synthetase–anticodon interaction. Small RNA substrates (minihelices, microhelices, and duplexes) are devoid of the anticodon triplet and may, therefore, be inefficiently aminoacylated because of a lack of anticodon-triggered acceptor stem distortion. To test this hypothesis, we constructed microhelices that vary in their ability to form a 1⋅72 base pair. The results of kinetic assays show that microhelix aminoacylation is activated by destabilization of this terminal base pair. The largest effect is seen when one of the two nucleotides of the pair is completely deleted. Activation of aminoacylation is also seen with the analogous deletion in a minihelix substrate for the closely related isoleucine enzyme. Thus, for at least the methionine and isoleucine systems, a built-in helix destabilization compensates in part for the lack of presumptive anticodon-induced acceptor stem distortion.

Assembly of a catalytic unit for RNA microhelix aminoacylation using nonspecific RNA binding domains

An assembly of a catalytic unit for aminoacylation of an RNA microhelix is demonstrated here. This assembly may recapitulate a step in the historical development of tRNA synthetases. The class-defining domain of a tRNA synthetase is closely related to the primordial enzyme that catalyzed synthesis of aminoacyl adenylate. RNA binding elements are imagined to have been added so that early RNA substrates could be docked proximal to the activated amino acid. RNA microhelices that recapitulate the acceptor stem of modern tRNAs are potential examples of early substrates. In this work, we examined a fragment of Escherichia coli alanyl-tRNA synthetase, which catalyzes aminoacyl adenylate formation but is virtually inactive for catalysis of RNA microhelix aminoacylation. Fusion to the fragment of either of two unrelated nonspecific RNA binding domains activated microhelix aminoacylation. Although the fusion proteins lacked the RNA sequence specificity of the natural enzyme, their activity was within 1–2 kcal⋅mol−1 of a truncated alanyl-tRNA synthetase that has aminoacylation activity sufficient to sustain cell growth. These results show that, starting with an activity for adenylate synthesis, barriers are relatively low for building catalytic units for aminoacylation of RNA helices.

The Bacillus subtilis crh gene encodes a HPr-like protein involved in carbon catabolite repression

Carbon catabolite repression (CCR) of several Bacillus subtilis catabolic genes is mediated by ATP-dependent phosphorylation of histidine-containing protein (HPr), a phosphocarrier protein of the phosphoenolpyruvate (PEP): sugar phosphotransferase system. In this study, we report the discovery of a new B. subtilis gene encoding a HPr-like protein, Crh (for catabolite repression HPr), composed of 85 amino acids. Crh exhibits 45% sequence identity with HPr, but the active site His-15 of HPr is replaced with a glutamine in Crh. Crh is therefore not phosphorylated by PEP and enzyme I, but is phosphorylated by ATP and the HPr kinase in the presence of fructose-1,6-bisphosphate. We determined Ser-46 as the site of phosphorylation in Crh by carrying out mass spectrometry with peptides obtained by tryptic digestion or CNBr cleavage. In a B. subtilis ptsH1 mutant strain, synthesis of β-xylosidase, inositol dehydrogenase, and levanase was only partially relieved from CCR. Additional disruption of the crh gene caused almost complete relief from CCR. In a ptsH1 crh1 mutant, producing HPr and Crh in which Ser-46 is replaced with a nonphosphorylatable alanyl residue, expression of β-xylosidase was also completely relieved from glucose repression. These results suggest that CCR of certain catabolic operons requires, in addition to CcpA, ATP-dependent phosphorylation of Crh, and HPr at Ser-46.