Two inducers of plant defense responses, 2,6-dichloroisonicotinec acid and salicylic acid, inhibit catalase activity in tobacco.

2,6-Dichloroisonicotinic acid (INA) and salicylic acid (SA) are potent inducers of plant defense responses including the synthesis of pathogenesis-related (PR) proteins and the development of enhanced disease resistance. A soluble SA-binding protein has been purified from tobacco with an affinity and specificity of binding that suggest it is a SA receptor. Recently, this protein has been shown to be a catalase whose enzymatic activity is inhibited by SA binding. We have proposed that the resulting increase in intracellular levels of reactive oxygen species plays a role in the induction of defense responses such as PR protein gene expression. Here we report that INA, like SA, binds the SA-binding protein/catalase and inhibits its enzymatic activity. In fact, the dose-response curves for inhibition of catalase by these two compounds are similar. Furthermore, the ability of both INA analogues and SA derivatives to bind and inhibit tobacco catalase correlates with their biological activity to induce PR-1 gene expression and enhance resistance to tobacco mosaic virus. Comparison of the structures of INA, SA, and their analogues reveals several common features that appear to be important for biological activity. Thus, these results not only suggest that INA and SA share the same mechanism of action that involves binding and inhibition of catalase but also further indicate an important role for reactive oxygen species in the induction of certain plant defense responses. This is supported by the demonstration that INA-mediated PR-1 gene activation is suppressed by antioxidants.

Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain.

According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation. In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death. To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria. PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death. In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta. Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis. These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease.

Augmented DNA-binding activity of p53 protein encoded by a carboxyl-terminal alternatively spliced mRNA is blocked by p53 protein encoded by the regularly spliced form.

DNA-binding activity of the wild-type p53 is central to its function in vivo. However, recombinant or in vitro translated wild-type p53 proteins, unless modified, are poor DNA binders. The fact that the in vitro produced protein gains DNA-binding activity upon modification at the C terminus raises the possibility that similar mechanisms may exist in the cell. Data presented here show that a C-terminal alternatively spliced wild-type p53 (ASp53) mRNA expressed by bacteria or transcribed in vitro codes for a p53 protein that efficiently binds DNA. Our results support the conclusion that the augmented DNA binding activity of an ASp53 protein is probably due to attenuation of the negative effect residing at the C terminus of the wild-type p53 protein encoded by the regularly spliced mRNA (RSp53) rather than acquisition of additional functionality by the alternatively spliced C' terminus. In addition, we found that ASp53 forms a complex with the non-DNA-binding RSp53, which in turn blocks the DNA-binding activity of ASp53. Interaction between these two wild-type p53 proteins may underline a mechanism that controls the activity of the wild-type p53 protein in the cell.

Direct measurement of oligonucleotide binding stoichiometry of gene V protein by mass spectrometry.

The binding stoichiometry of gene V protein from bacteriophage f1 to several oligonucleotides was studied using electrospray ionization-mass spectrometry (ESI-MS). Using mild mass spectrometer interface conditions that preserve noncovalent associations in solution, gene V protein was observed as dimer ions from a 10 mM NH4OAc solution. Addition of oligonucleotides resulted in formation of protein-oligonucleotide complexes with stoichiometry of approximately four nucleotides (nt) per protein monomer. A 16-mer oligonucleotide gave predominantly a 4:1 (protein monomer: oligonucleotide) complex while oligonucleotides shorter than 15 nt showed stoichiometries of 2:1. Stoichiometries and relative binding constants for a mixture of oligonucleotides were readily measured using mass spectrometry. The binding stoichiometry of the protein with the 16-mer oligonucleotide was measured independently using size-exclusion chromatography and the results were consistent with the mass spectrometric data. These results demonstrate, for the first time, the observation and stoichiometric measurement of protein-oligonucleotide complexes using ESI-MS. The sensitivity and high resolution of ESI-MS should make it a useful too] in the study of protein-DNA interactions.

Automated de novo sequencing of proteins by tandem high-resolution mass spectrometry

A de novo sequencing program for proteins is described that uses tandem MS data from electron capture dissociation and collisionally activated dissociation of electrosprayed protein ions. Computer automation is used to convert the fragment ion mass values derived from these spectra into the most probable protein sequence, without distinguishing Leu/Ile. Minimum human input is necessary for the data reduction and interpretation. No extra chemistry is necessary to distinguish N- and C-terminal fragments in the mass spectra, as this is determined from the electron capture dissociation data. With parts-per-million mass accuracy (now available by using higher field Fourier transform MS instruments), the complete sequences of ubiquitin (8.6 kDa) and melittin (2.8 kDa) were predicted correctly by the program. The data available also provided 91% of the cytochrome c (12.4 kDa) sequence (essentially complete except for the tandem MS-resistant region K13–V20 that contains the cyclic heme). Uncorrected mass values from a 6-T instrument still gave 86% of the sequence for ubiquitin, except for distinguishing Gln/Lys. Extensive sequencing of larger proteins should be possible by applying the algorithm to pieces of ≈10-kDa size, such as products of limited proteolysis.

Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3′→5′ exonuclease activity

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3′→5′ DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

Mechanism for nucleic acid chaperone activity of HIV-1 nucleocapsid protein revealed by single molecule stretching

The nucleocapsid protein (NC) of HIV type 1 is a nucleic acid chaperone that facilitates the rearrangement of nucleic acids into conformations containing the maximum number of complementary base pairs. We use an optical tweezers instrument to stretch single DNA molecules from the helix to coil state at room temperature in the presence of NC and a mutant form (SSHS NC) that lacks the two zinc finger structures present in NC. Although both NC and SSHS NC facilitate annealing of complementary strands through electrostatic attraction, only NC destabilizes the helical form of DNA and reduces the cooperativity of the helix-coil transition. In particular, we find that the helix-coil transition free energy at room temperature is significantly reduced in the presence of NC. Thus, upon NC binding, it is likely that thermodynamic fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations to find the lowest energy state. The reduced cooperativity allows these fluctuations to occur in the middle of complex double-stranded structures. The reduced stability and cooperativity, coupled with the electrostatic attraction generated by the high charge density of NC, is responsible for the nucleic acid chaperone activity of this protein.

Manipulation of Catalase Levels Produces Altered Photosynthesis in Transgenic Tobacco Plants1

Constructs containing the cDNAs encoding the primary leaf catalase in Nicotiana or subunit 1 of cottonseed (Gossypium hirsutum) catalase were introduced in the sense and antisense orientation into the Nicotiana tabacum genome. The N. tabacum leaf cDNA specifically overexpressed CAT-1, the high catalytic form, activity. Antisense constructs reduced leaf catalase specific activities from 0.20 to 0.75 times those of wild type (WT), and overexpression constructs increased catalase specific activities from 1.25 to more than 2.0 times those of WT. The NADH-hydroxypyruvate reductase specific activity in transgenic plants was similar to that in WT. The effect of antisense constructs on photorespiration was studied in transgenic plants by measuring the CO2 compensation point (Γ) at a leaf temperature of 38°C. A significant linear increase was observed in Γ with decreasing catalase (at 50% lower catalase activity Γ increased 39%). There was a significant temperature-dependent linear decrease in Γ in transgenic leaves with elevated catalase compared with WT leaves (at 50% higher catalase Γ decreased 17%). At 29°C, Γ also decreased with increasing catalase in transgenic leaves compared with WT leaves, but the trend was not statistically significant. Rates of dark respiration were the same in WT and transgenic leaves. Thus, photorespiratory losses of CO2 were significantly reduced with increasing catalase activities at 38°C, indicating that the stoichiometry of photorespiratory CO2 formation per glycolate oxidized normally increases at higher temperatures because of enhanced peroxidation.

Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress.

Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress.

Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.

Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells. A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM. Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively. ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen). The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively. These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action.

Developmentally related responses of maize catalase genes to salicylic acid.

The response of the maize catalase genes (Cat1, Cat2, and Cat3) to salicylic acid (SA) was examined at two distinct developmental stages: embryogenesis and germination. A unique, germination-related differential response of each maize catalase gene to various doses of SA was observed. During late embryogenesis, total catalase activity in scutella increased dramatically with 1 mM SA treatment. The accumulation of Cat2 transcript and CAT-2 isozyme protein provided the major contribution to the observed increase in total catalase activity. This increase was paralleled by the enhanced growth of germinated embryos at that stage. In a CAT-2 null mutant line, a full compensation of total catalase activity by the CAT-1 isozyme was observed in the presence of SA. This suggests that catalase is important for maintenance of normal cellular processes under stress conditions. SA at 1 mM, which enhances growth of precociously germinated embryos, appeared to inhibit seed germination at 1 day after inhibition. Furthermore, Cat2 transcript accumulation was inhibited at this stage. SA is probably not a direct signal for the induction of the catalase genes. Other signals, possibly germination-related regulator(s), might be responsible for the induction of the catalase genes. The effect of SA on the activity of purified catalase protein was also examined.

Non-p53 p53RE binding protein, a human transcription factor functionally analogous to P53

The transactivation activity of the p53 tumor suppressor protein is critical for regulating cell growth and apoptosis. We describe the identification of a transcription factor that is functionally similar to p53 and contains the same DNA binding and transcription activities specific for the p53 responsive DNA element (p53RE). This protein was highly purified through chromatography from HeLa cell extracts. The purified protein was able to bind specifically to the p53RE derived from a p21waf1 promoter and to stimulate p53RE-dependent transcription but not basal transcription in vitro. Its DNA-binding activity was inhibited by the wild type but not mutant p53RE-containing DNA oligomers. Also, this p53RE-binding activity was found in human p53 null Saos-2 osteosarcoma and H1299 small cell lung carcinoma cells. Interestingly, this activity exhibited a p53RE sequence preference that was distinct from the p53 protein. The activity is neither p53 nor p73, because anti-p53 or anti-73 antibodies were unable to detect this purified protein nor were the antibodies able to alter the p53-like activity, the p53RE-protein complex. These results demonstrate that, besides p73, an additional p53-like protein exists in cells, which is named NBP for non-p53, p53RE binding protein.

Adeno-associated viral-mediated catalase expression suppresses optic neuritis in experimental allergic encephalomyelitis

Suppression of oxidative injury by viral-mediated transfer of the human catalase gene was tested in the optic nerves of animals with experimental allergic encephalomyelitis (EAE). EAE is an inflammatory autoimmune disorder of primary central nervous system demyelination that has been frequently used as an animal model for the human disease multiple sclerosis (MS). The optic nerve is a frequent site of involvement common to both EAE and MS. Recombinant adeno-associated virus containing the human gene for catalase was injected over the right optic nerve heads of SJL/J mice that were simultaneously sensitized for EAE. After 1 month, cell-specific catalase activity, evaluated by quantitation of catalase immunogold, was increased approximately 2-fold each in endothelia, oligodendroglia, astrocytes, and axons of the optic nerve. Effects of catalase on the histologic lesions of EAE were measured by computerized analysis of the myelin sheath area (for demyelination), optic disc area (for optic nerve head swelling), extent of the cellular infiltrate, extravasated serum albumin labeled by immunogold (for blood–brain barrier disruption), and in vivo H2O2 reaction product. Relative to control, contralateral optic nerves injected with the recombinant virus without a therapeutic gene, catalase gene inoculation reduced demyelination by 38%, optic nerve head swelling by 29%, cellular infiltration by 34%, disruption of the blood–brain barrier by 64%, and in vivo levels of H2O2 by 61%. Because the efficacy of potential treatments for MS are usually initially tested in the EAE animal model, this study suggests that catalase gene delivery by using viral vectors may be a therapeutic strategy for suppression of MS.

Nup93, a Vertebrate Homologue of Yeast Nic96p, Forms a Complex with a Novel 205-kDa Protein and Is Required for Correct Nuclear Pore Assembly

Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human, rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAAO225, present in the human database. The putative human nucleoporin of 205 kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. To analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex–depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates.

The GTPase activating factor for transducin in rod photoreceptors is the complex between RGS9 and type 5 G protein β subunit

Proteins of the regulators of G protein signaling (RGS) family modulate the duration of intracellular signaling by stimulating the GTPase activity of G protein α subunits. It has been established that the ninth member of the RGS family (RGS9) participates in accelerating the GTPase activity of the photoreceptor-specific G protein, transducin. This process is essential for timely inactivation of the phototransduction cascade during the recovery from a photoresponse. Here we report that functionally active RGS9 from vertebrate photoreceptors exists as a tight complex with the long splice variant of the G protein β subunit (Gβ5L). RGS9 and Gβ5L also form a complex when coexpressed in cell culture. Our data are consistent with the recent observation that several RGS proteins, including RGS9, contain G protein γ-subunit like domain that can mediate their association with Gβ5 (Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S. F., Wall, M. A., Chung, S., Mangion, J., Arya, S., Gilman, A. G. & Siderovski, D. P. (1998) Proc. Natl. Acad. Sci. USA 95, 13307–13312). We report an example of such a complex whose cellular localization and function are clearly defined.