Identification of nuclear target proteins for S-nitrosylation in pathogen-treated Arabidopsis thaliana cell cultures.

Nitric oxide (NO) is a significant signalling molecule involved in the regulation of many different physiological processes in plants. One of the most imperative regulatory modes of action of NO is protein S-nitrosylation--the covalent attachment of an NO group to the sulfur atom of cysteine residues. In this study, we focus on S-nitrosylation of Arabidopsis nuclear proteins after pathogen infection. After treatment of Arabidopsis suspension cell cultures with pathogens, nuclear proteins were extracted and treated with the S-nitrosylating agent S-nitrosoglutathione (GSNO). A biotin switch assay was performed and biotin-labelled proteins were purified by neutravidin affinity chromatography and identified by mass spectrometry. A total of 135 proteins were identified, whereas nuclear localization has been described for 122 proteins of them. 117 of these proteins contain at least one cysteine residue. Most of the S-nitrosylated candidates were involved in protein and RNA metabolism, stress response, and cell organization and division. Interestingly, two plant-specific histone deacetylases were identified suggesting that nitric oxide regulated epigenetic processes in plants. In sum, this work provides a new collection of targets for protein S-nitrosylation in Arabidopsis and gives insight into the regulatory function of NO in the nucleus during plant defense response. Moreover, our data extend the knowledge on the regulatory function of NO in events located in the nucleus.

Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction.

BACKGROUND: Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg2+ in its activity was not studied in sufficient details. RESULTS: In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg2+/Mn2+, and that neither Zn2+ or nor Ca2+ can activate hTDP2. Mg2+ also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg2+ controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg2+ concentration above which it is inhibitory for hTDP2 activity. CONCLUSION: These results altogether reveal the optimal Mg2+ requirement in hTDP2 mediated reaction.

Extraction of nuclear proteins from root meristematic cells.

Fractionation and extraction of nuclear proteins are techniques intended to facilitate dedicated plant proteomic studies. These techniques rely on subcellular fractionation, which makes it possible to define and characterize the proteome of a subcellular organelle, in this case the cell nucleus. Nuclear protein fractionation is proposed as a method to be carried out according to the solubility of proteins in buffers of increasing ionic strength. This physical criterion, accompanied in some steps by the use of additional reagents, such as detergents or enzymes, produces fractions that have been demonstrated to have functional significance. The proposed procedure yields five fractions, the first of them containing proteins associated with the nuclear envelope and remnants of the cytoskeleton. The second fraction, which is soluble at low ionic strength, contains ribonucleoproteins active in nuclear RNA metabolism. After increasing ionic strength and digesting with DNase, the result is the chromatin fraction. Finally, the fourth and fifth fractions correspond to the nuclear matrix and are obtained, respectively, by solubilization in high salt concentration and in the form of the residual pellet, which is only soluble in 7 M urea under sonication. This procedure offers a wide range of applicability, even in the cases in which the genome of the particular species investigated is not sequenced. In general, the functional criteria driving the extraction method described here will make this method capable of generating valuable and useful information.

CAR expression and inducibility of CYP2B genes in liver of rats treated with PB-like inducers.

The expression of the CAR gene and inducibility of CYP2B protein in the liver of male Wistar rats treated with phenobarbital (PB) and triphenyldioxane (TPD) were investigated. To clarify the role of phosphorylation/dephosphorylation in these processes, rats were treated with inhibitors of Ca(2+)/calmodulin-dependent kinase II (W7) or protein phosphatases PP1 and PP2A (OA) before induction. Constitutive expression of the CAR gene in livers of untreated rats was detected by multiplex RT-PCR. Treatment with W7 resulted in a 2.8-fold induction of CAR gene expression, whereas OA led to a 2.4-fold decrease of the mRNA level. The same results were obtained for CYP2B genes expression, which were increased by W7 treatment (two-fold) and decreased by OA (2.3-fold). PB-induction did not lead to significant alteration in the level of CAR gene expression, although CYP2B genes expression was enhanced two-fold over control values. TPD caused a two-fold increase of both CAR and CYP2B mRNA levels. Both inducers reduced the effects of inhibitors on CAR gene expression. Results of EMSA showed that PB, TPD or W7 alone induced formation of complexes of NR1 with nuclear proteins. Appearance of the complexes correlated with an increase in CYP2B expression, and their intensities were modulated by the protein kinase inhibitors. Thus, our results demonstrate that constitutive expressions of CAR as well as CYP2B during induction are regulated by phosphorylation/dephosphorylation processes.

Purification, crystallization and preliminary X-ray analysis of the N-terminal domain of NO38, a nucleolar protein from Xenopus laevis.

NO38 is a multidomain protein that belongs to the nucleoplasmin (Np) family. Previous studies have suggested that acidic chaperones such as Np may function as histone-storage platforms. Here, the purification and crystallization of the N-terminal domain of NO38 in two crystal forms is reported. The C2 crystal form diffracts to 1.9 A and contains two pentamers in the asymmetric unit, while the P1 crystals diffract to 1.7 A and contain a non-crystallographic decamer with 522 symmetry. By analogy with Np, the NO38 decamer may represent the active form of this chaperone.

Characterization and chondrocyte differentiation stage-specific expression of KRAB zinc-finger protein gene ZNF470.

As part of a study to identify novel transcriptional regulators of chondrogenesis-related gene expression, we have cloned and characterized cDNA for zinc-finger protein 470 (ZNF470), the human ortholog of which encodes a 717 amino acid residue protein containing 17 Cys(2)His(2) zinc-finger domains, as well as KRAB-A and KRAB-B motifs. The cDNA library used to isolate the initial ZNF470 clone was prepared from human bone marrow-derived mesenchymal progenitor cells at an intermediate stage of chondrogenic differentiation. We have determined the intron-exon structure of the human ZNF470 gene, which has been mapped to a zinc-finger cluster in a known imprinted region of human chromosome 19q13.4. ZNF470 is expressed at high levels in human testis and is expressed at low or undetectible levels in other adult tissues. Human ZNF470 expressed in mammalian cells as an EGFP fusion protein localizes predominantly to the nucleus, consistent with a role in transcriptional regulation. ZNF470, analyzed by quantitative real time PCR, was transiently expressed before the maximal expression of COL2A1 during chondrogenic differentiation in vitro. We have also characterized the bovine ortholog of human ZNF470, which encodes a 508 amino acid residue protein having 10 zinc-finger domains. A bovine ZNF470 cDNA clone was used to examine expression of ZNF470 in bovine articular chondrocytes treated with retinoic acid to stimulate dedifferentiation. Bovine ZNF470 expression was undetectable in freshly isolated bovine articular chondrocytes, but was dramatically upregulated in dedifferentiated retinoic acid-treated chondrocytes. These results, in two model systems, suggest a possible role for ZNF470 in the regulation of chondrogenesis-specific gene expression.

Prostate specific antigen expression is down-regulated by selenium through disruption of androgen receptor signaling.

A previous controlled intervention trial showed that selenium supplementation was effective in reducing the incidence of prostate cancer. Physiological concentrations of selenium have also been reported to inhibit the growth of human prostate cancer cells in vitro. The present study describes the observation that selenium was able to significantly down-regulate the expression of prostate-specific antigen (PSA) transcript and protein within hours in the androgen-responsive LNCaP cells. Decreases in androgen receptor (AR) transcript and protein followed a similar dose and time response pattern upon exposure to selenium. The reduction of AR and PSA expression by selenium occurred well before any significant change in cell number. With the use of a luciferase reporter construct linked to either the PSA promoter or the androgen responsive element, it was found that selenium inhibited the trans-activating activity of AR in cells transfected with the wild-type AR expression vector. Selenium also suppressed the binding of AR to the androgen responsive element site, as evidenced by electrophoretic mobility shift assay of the AR-androgen responsive element complex. In view of the fact that PSA is a well-accepted prognostic indicator of prostate cancer, an important implication of this study is that a selenium intervention strategy aimed at toning down the amplitude of androgen signaling could be helpful in controlling morbidity of this disease.

Purification and characterization of human nucleolar phosphoprotein 140 expressed in Escherichia coli.

Human nucleolar phosphoprotein 140, hNopp140, is one of the most highly phosphorylated mammalian proteins, which is involved in the biogenesis of nucleolus. It regulates the transcription of rDNA and has a tendency to bind to doxorubicin, which is widely used as an anti-cancer drug. The biochemical and biophysical property of hNopp140 has not been reported due to the fact that it is rather difficult to obtain protein in large enough quantity. In this paper, we report the cloning and overexpression of the soluble form of hNopp140 in Escherichia coli. The protein was purified to more than 90% homogeneity using hydroxyapatite and ion exchange chromatography. The purified protein can be extensively phosphorylated by casein kinase II and oligomerized into an insoluble aggregate in the presence of magnesium, carbonate, and fluoride ions.

The auxin-induced maize gene ZmSAUR2 encodes a short-lived nuclear protein expressed in elongating tissues.

By differential screening of a cDNA library from auxin-induced maize coleoptiles we have isolated and characterized a SAUR gene, designated ZmSAUR2, belonging to a not yet characterized subtype of the SAUR family. ZmSAUR2 encodes a 15.3-kDa protein and is specifically induced by auxin in elongating coleoptile tissue but not in primary leaves or in roots. The transcript level rapidly increased within minutes and preceded auxin-stimulated elongation of coleoptile segments. Cycloheximide also induced ZmSAUR2 transcription, as has been shown for other early auxin-induced genes, whereas abscisic acid, brassinolide, ethylene, gibberellic acid, kinetin, and methyl jasmonate did not provoke an increase in ZmSAUR2 mRNA abundance. In pulse-chase experiments using auxin-induced coleoptiles and an anti-ZmSAUR2 antibody we were able to precipitate a protein of the expected molecular mass and to determine a half-life of about 7 min, which is among the shortest known in eukaryotes. In gel shift assays binding of calmodulin to ZmSAUR2 was demonstrated, suggesting the possibility of post-transcriptional regulation. Upon transformation of onion epidermal cells with a ZmSAUR2::GUS construct the corresponding chimeric protein was detected in the nucleus. The results suggest that ZmSAUR2 encodes a short-lived nuclear protein that might be involved in auxin-mediated cell elongation.

PBF-2 is a novel single-stranded DNA binding factor implicated in PR-10a gene activation in potato.

Elicitor-induced activation of the potato pathogenesis-related gene PR-10a requires a 30-bp promoter sequence termed the ERE (elicitor response element) that is bound by the nuclear factor PBF-2 (PR-10a binding factor 2). In this study, PBF-2 has been purified to near homogeneity from elicited tubers through a combination of anion-exchange and DNA affinity chromatography. Evidence demonstrates that inactive PBF-2 is stored in the nuclei of fresh tubers and becomes available for binding to the ERE upon elicitation. A protein with an apparent molecular mass of 24 kD (p24) is a DNA binding component of PBF-2. A cDNA encoding p24 has been cloned and encodes a novel protein with a potential transcriptional activation domain that could also act as a single-stranded DNA binding domain. Both PBF-2 and the cDNA-encoded protein bind with high affinity to the single-stranded form of the ERE in a sequence-specific manner. The inverted repeat sequence of the ERE, TGACAnnnnTGTCA, is critical for binding of this factor in vitro and for PR-10a expression in vivo, supporting the role of PBF-2 as a transcriptional regulator.

Integrated hepatitis B virus DNA preserves the binding sequence of transcription factor Yin and Yang 1 at the virus-cell junction.

Accumulated findings have indicated that hepatitis B virus (HBV) DNA integrates into the cellular DNA of HBV-infected chronic hepatitis tissues. The integrated sequence (IS) of HBV DNA at the virus-cell junction is conserved in a 25-bp region which is adjacent to direct repeat 1. A cellular protein which we purified from the nuclear extract of HepG2 cells binds to the IS and was designated IS binding protein 3 (ISBP3). The amino acid sequence of ISBP3 was determined and found to be identical to that of transcription initiation factor Yin and Yang 1 (YY1). An antibody against C-terminal amino acids of YY1 recognized ISBP3 in a Western blot analysis and an electrophoretic mobility shift assay. Furthermore, ISBP3 also interacted with Y3, which corresponds to the YY1 binding sequence, to enhance intramolecular recombination of polyomavirus DNA. Although YY1 is known as a transcription factor, the IS did not exhibit any effect on the transcription of precore and pregenome RNAs. The possible involvement of YY1 in the intramolecular recombination of linear replicative HBV DNA has been examined (Y. Hayashi et al., unpublished data). Data suggest that YY1 is involved in the joining reaction between HBV DNA and cellular DNA to form the virus-cell junction.

Identification of chURP, a nuclear calmodulin-binding protein related to hnRNP-U.

In a screen for myosin-like proteins in embryonic chicken brain, we have identified a novel nuclear protein structurally related to hnRNP-U (heterogeneous nuclear ribonuclear protein U). We have called this protein chURP, for chicken U-related protein. In this screen, chURP was immunoreactive with two myosin antibodies and, in common with the unconventional myosins, bound calmodulin in vitro in both the presence and absence of calcium ions. Determination of 757 amino acids of the chURP sequence revealed that it shares 41% amino acid identity with human and rat hnRNP-U, although chURP and hnRNP-U appear not to be orthologous proteins. ChURP is ubiquitously expressed in the nuclei of all chick tissues and, as one of a growing number of calmodulin-binding proteins to be identified in the nucleus, further highlights the potential of calmodulin as a regulator of nuclear metabolism.

ETO-2, a new member of the ETO-family of nuclear proteins.

The t(8;21) is associated with 12-15% of acute myelogenous leukemias of the M2 subtype. The translocation results in the fusion of two genes, AML1 (CBFA2) on chromosome 21 and ETO (MTG8) on chromosome 8. AML1 encodes a DNA binding factor; the ETO protein product is less well characterized, but is thought to be a transcription factor. Here we describe the isolation and characterization of ETO-2, a murine cDNA that encodes a new member of the ETO family of proteins. ETO-2 is 75% identical to murine ETO and shares very high sequence identities over four regions of the protein with ETO (domain I-III and zinc-finger). Northern analysis identifies ETO-2 transcripts in many of the murine tissues analysed and in the developing mouse embryo. ETO-2 is also expressed in myeloid and erythroid cell lines. We confirmed the nuclear localization of ETO-2 and demonstrated that domain III and the zinc-finger region are not required for nuclear localization. We further showed that a region within ETO, containing domain II, mediates dimerization among family members. This region is conserved in the oncoprotein AML-1/ETO. The recent identification of another ETO-like protein, myeloid translocation gene-related protein 1, together with the data presented here, demonstrates that at least three ETO proteins exist with the potential to form dimers in the cell nucleus.

Isolation, tissue expression, and chromosomal assignment of a novel human gene which encodes a protein with RING finger motif.

We identified a novel gene encoding a RING finger (C3HC4-type zinc finger) protein from a human neuroblastoma full-length enriched cDNA library. This cDNA clone consists of 1919 nucleotides with an open reading frame of a 485-amino acid protein. From reverse transcription (RT)-polymerase chain reaction (PCR) analysis, the messenger RNA was ubiquitously expressed in various human adult tissues. The chromosomal location of the gene was determined on the chromosome 6p21.3 region by PCR-based analyses with both a human/rodent monochromosomal hybrid cell panel and a radiation hybrid mapping panel.

Isolation and characterization of a novel coactivator protein, NCoA-62, involved in vitamin D-mediated transcription.

The vitamin D receptor (VDR) forms a heterodimeric complex with retinoid X receptor (RXR) and binds to vitamin D-responsive promoter elements to regulate the transcription of specific genes or gene networks. The precise mechanism of transcriptional regulation by the VDR.RXR heterodimer is not well understood, but it may involve interactions of VDR.RXR with transcriptional coactivator or corepressor proteins. Here, a yeast two-hybrid strategy was used to isolate proteins that selectively interacted with VDR and other nuclear receptors. One cDNA clone designated NCoA-62, encoded a 62, 000-Da protein that is highly related to BX42, a Drosophila melanogaster nuclear protein involved in ecdysone-stimulated gene expression. Yeast two-hybrid studies and in vitro protein-protein interaction assays using glutathione S-transferase fusion proteins demonstrated that NCoA-62 formed a direct protein-protein contact with the ligand binding domain of VDR. Coexpression of NCoA-62 in a vitamin D-responsive transient gene expression system augmented 1, 25-dihydroxyvitamin D3-activated transcription, but it had little or no effect on basal transcription or gal4-VP16-activated transcription. NCoA-62 also interacted with retinoid receptors, and its expression enhanced retinoic acid-, estrogen-, and glucocorticoid-mediated gene expression. These data indicate that NCoA-62 may be classified into an emerging set of transcriptional coactivator proteins that function to facilitate vitamin D- and other nuclear receptor-mediated transcriptional pathways.

Characterization of a human RPD3 ortholog, HDAC3.

Histone acetylation levels in cells result from a dynamic equilibrium between competing histone acetylases and deacetylases. Changes in histone acetylation levels occur during both transcriptional activation and silencing. Cloning of the cDNA for a human histone deacetylase (HDAC1) has shown that it represents a human ortholog of the yeast transcriptional regulator RPD3. We have screened the expressed sequence tag database (National Center for Biotechnology Information) with the yeast RPD3 sequence and identified a human ortholog of RPD3, HDAC3. This cDNA encodes a protein of 428 amino acids with 58% sequence identity with HDAC1p. By using a specific polyclonal antiserum recognizing the C-terminal domain of HDAC3p and Western blotting, we detected a single approximately 49-kDa band in several tumor cell lines. HDAC3p is expressed predominantly in the nuclear compartment. Immunoprecipitation experiments with either an antiserum against HDAC3p or an anti-FLAG antiserum and a flagged HDAC3 cDNA showed that HDAc3p exhibits deacetylase activity both on free histones and on purified nucleosomes. This deacetylase activity is inhibited by trichostatin, trapoxin, and butyrate in vitro to the same degree as the deacetylase activity associated to HDAC1p. These observations identify another member of a growing family of human HDAC genes.

The retinoblastoma interacting zinc finger gene RIZ produces a PR domain-lacking product through an internal promoter.

The PR domain is a newly recognized protein motif that characterizes a subfamily of Krüppel-like zinc finger genes. Members of the PR domain family have been shown to play important roles in cell differentiation and malignant transformation. The RIZ gene is the founding member of this family; it was isolated because its gene products can bind to the retinoblastoma tumor suppressor protein. Here, we have studied the RIZ gene structure and expression. By immunoprecipitation and immunoblot analysis we identified two different RIZ protein products of 280 and 250 kDa, designated RIZ1 and RIZ2, respectively. The 280-kDa RIZ1 product comigrated with the RIZ cDNA-derived polypeptide. The 250-kDa RIZ2 product lacked the NH2-terminal PR domain of RIZ1; it comigrated with a truncated RIZ1 polypeptide that was initiated from an internal ATG codon. Both the full-length and the truncated RIZ1 polypeptide were located in the nucleus as shown by transfection and immunofluorescence analysis. We identified the RIZ2 transcripts and showed that they were produced by an internal promoter located at the 5' boundary of coding exon 5. RNase protection analysis revealed similar ratios of RIZ1 and RIZ2 transcripts in most adult rat tissues except in testis, where RIZ1 was more abundant than RIZ2. These observations were strikingly similar to those described for the MDS1-EVI1 cancer gene, which also normally gives rise to a PR domain-lacking product, EVI1, because of an internal promoter.

PICK1: a perinuclear binding protein and substrate for protein kinase C isolated by the yeast two-hybrid system.

Protein kinase C (PKC) plays a central role in the control of proliferation and differentiation of a wide range of cell types by mediating the signal transduction response to hormones and growth factors. Upon activation by diacylglycerol, PKC translocates to different subcellular sites where it phosphorylates numerous proteins, most of which are unidentified. We used the yeast two-hybrid system to identify proteins that interact with activated PKC alpha. Using the catalytic region of PKC fused to the DNA binding domain of yeast GAL4 as "bait" to screen a mouse T cell cDNA library in which cDNA was fused to the GAL4 activation domain, we cloned several novel proteins that interact with C-kinase (PICKs). One of these proteins, designated PICK1, interacts specifically with the catalytic domain of PKC and is an efficient substrate for phosphorylation by PKC in vitro and in vivo. PICK1 is localized to the perinuclear region and is phosphorylated in response to PKC activation. PICK1 and other PICKs may play important roles in mediating the actions of PKC.

Relief of YY1-induced transcriptional repression by protein-protein interaction with the nucleolar phosphoprotein B23.

Previous studies have shown that the transcription factor YY1 can both activate and repress transcription of many mammalian genes (reviewed by Hahn (Hahn, S. (1992) Curr. Biol. 2, 152-154)). Given the diverse effects of the YY1 protein, it seems likely that its function depends on interaction with other cellular factors. We have used the yeast two-hybrid system to isolate mouse cDNAs encoding proteins capable of directly binding to YY1. Sequence analysis of one clone revealed it had an open reading frame with the potential to code for a protein nearly identical to the previously published mouse nucleolar phosphoprotein B23. The YY1.B23 complex is specific, and occurs in vivo and in vitro. Overexpression of the B23 protein can reverse the transcriptional repression exerted by YY1. These results suggest a role for a nucleolar protein as a component in transcription and provide a possible mechanism for transcriptional regulation by YY1.

Cloning and characterization of AKAP 95, a nuclear protein that associates with the regulatory subunit of type II cAMP-dependent protein kinase.

The subcellular location of the type II cAMP-dependent protein kinase is dictated by the interaction of the regulatory subunit (RII) with A-kinase anchor proteins (AKAPs). Using an interaction cloning strategy with RII alpha as a probe, we have isolated cDNAs encoding a novel 761-amino acid protein (named AKAP 95) that contains both RII- and DNA-binding domains. Deletion analysis and peptide studies revealed that the RII-binding domain of AKAP 95 is located between residues 642 and 659 and includes a predicted amphipathic helix. Zinc overlay and DNA binding studies suggest that the DNA-binding domain is composed of two CC/HH-type zinc fingers between residues 464 and 486 and residues 553 and 576. The AKAP was detected in a nuclear matrix fraction, and immunofluorescence using purified anti-AKAP 95 antibodies revealed a distinct nuclear staining in a variety of cell types. Direct overlay of fluorescein isothiocyanate-labeled RII alpha onto fixed rat embryo fibroblasts showed that high-affinity binding sites for RII exist in the nucleus and that these sites are blocked by an anchoring inhibitor peptide. Furthermore, AKAP 95 was detected in preparations of RII that were purified from cellular extracts using cAMP-agarose. The results suggest that AKAP 95 could play a role in targeting type II cAMP-dependent protein kinase for cAMP-responsive nuclear events.