Cambial meristem dormancy in trees involves extensive remodelling of the transcriptome

The establishment of the dormant state in meristems involves considerable physiological and metabolic alterations necessary for surviving unfavourable growth conditions. However, a global molecular analysis of dormancy in meristems has been hampered by the difficulty in isolating meristem cells. We used cryosectioning to isolate purified cambial meristem cells from the woody plant Populus tremula during active growth and dormancy. These samples were used to generate meristem-specific cDNA libraries and for cDNA microarray experiments to define the global transcriptional changes underlying cambial dormancy. The results indicate a significant reduction in the complexity of the cambial transcriptome in the dormant state. Although cell division is terminated in the dormant cambium, the cell cycle machinery appears to be maintained in a skeletal state as suggested by the continued presence of transcripts for several cell cycle regulators. The downregulation of PttPIN1 and PttPIN2 transcripts explains the reduced basipetal polar auxin transport during dormancy. The induction of a member of the SINA family of ubiquitin ligases implicated in auxin signalling indicates a potential mechanism for modulation of auxin sensitivity during cambial dormancy. The metabolic alterations during dormancy are mirrored in the induction of genes involved in starch breakdown and the glyoxysomal cycle. Interestingly, the induction of RGA1 like gene suggests modification of gibberellin signalling in cambial dormancy. The induction of genes such as poplar orthologues of FIE and HAP2 indicates a potential role for these global regulators of transcription in orchestrating extensive changes in gene expression during dormancy.

Nedd4-2 functionally interacts with ClC-5 - Involvement in constitutive albumin endocytosis in proximal tubule cells

Constitutive albumin uptake by the proximal tubule is achieved by a receptor-mediated process in which the Cl- channel, ClC-5, plays an obligate role. Here we investigated the functional interaction between ClC-5 and ubiquitin ligases Nedd4 and Nedd4-2 and their role in albumin uptake in opossum kidney proximal tubule (OK) cells. In vivo immunoprecipitation using an anti-HECT antibody demonstrated that ClC-5 bound to ubiquitin ligases, whereas glutathione S-transferase pull-downs confirmed that the C terminus of ClC-5 bound both Nedd4 and Nedd4-2. Nedd4-2 alone was able to alter ClC-5 currents in Xenopus oocytes by decreasing cell surface expression of ClC-5. In OK cells, a physiological concentration of albumin (10 mug/ml) rapidly increased cell surface expression of ClC-5, which was also accompanied by the ubiquitination of ClC-5. Albumin uptake was reduced by inhibiting either the lysosome or proteasome. Total levels of Nedd4-2 and proteasome activity also increased rapidly in response to albumin. Overexpression of ligase defective Nedd4-2 or knockdown of endogenous Nedd4-2 with small interfering RNA resulted in significant decreases in albumin uptake. In contrast, pathophysiological concentrations of albumin (100 and 1000 mug/ml) reduced the levels of ClC-5 and Nedd4-2 and the activity of the proteasome to the levels seen in the absence of albumin. These data demonstrate that normal constitutive uptake of albumin by the proximal tubule requires Nedd4-2, which may act via ubiquitination to shunt ClC-5 into the endocytic pathway.

Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2

Nedd4 and Nedd4-2 are ubiquitin-protein ligases known to regulate a number of membrane proteins including receptors and ion transporters. Regulation of the epithelial Na+ channel by Nedd4 and Nedd4-2 is mediated via interactions between the PY motifs of the epithelial sodium channel subunits and the Nedd4/Nedd4-2 WW domains. This example serves as a model for the regulation of other PY motif-containing ion channels by Nedd4 and Nedd4-2. We found that the carboxyl termini of the six voltage-gated Na+ (Na-v) channels contain typical PY motifs (PPXY), and a further Na-v contains a PY motif variant (LPXY). Not only did we demonstrate by Far-Western analysis that Nedd4 and Nedd4-2 interact with the PY motif-containing Na-v channels, but we also showed that these channels have conserved WW domain binding specificity. We further showed that the carboxyl termini fusion proteins of one central nervous system and one peripheral nervous system-derived Na+ channel (Na(v)1.2 and Na(v)1.7, respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes, Nedd4-2 strongly inhibited the activities of all three Na(v)s (Na(v)1.2, Na(v)1.7, and Na(v)1.8) tested. Interestingly, Nedd4 suppressed the activity of Na(v)1.2 and Na(v)1.7 but was a poor inhibitor of Na(v)1.8. Our results provide evidence that Nedd4 and Nedd4-2 are likely to be key regulators of specific neuronal Na-v channels in vivo.

N4WBP5, a potential target for ubiquitination by the Nedd4 family of proteins, is a novel golgi-associated protein

Nedd4 belongs to a family of ubiquitin-protein ligases that is characterized by 2-4 WW domains, a carboxyl-terminal Hect ((h) under bar omologous to (E) under bar6-AP (C) under bar arboxyl (t) under bar erminus)-domain and in most cases an amino-terminal C2 domain. We had previously identified a series of proteins that associates with the WW domains of Nedd4. In this paper, we demonstrate that one of the Nedd4-binding proteins, N4WBP5, belongs to a small group of evolutionarily conserved proteins with three transmembrane domains. N4WBP5 binds Nedd4 WW domains via the two PPXY motifs present in the amino terminus of the protein. In addition to Nedd4, N4WBP5 can interact with the WW domains of a number of Nedd4 family members and is ubiquitinated. Endogenous N4WBP5 localizes to the Golgi complex. Ectopic expression of the protein disrupts the structure of the Golgi, suggesting that N4WBP5 forms part of a family of integral Golgi membrane proteins. Based on previous observations in yeast, we propose that N4WBP5 may act as an adaptor for Nedd4-like proteins and their putative targets to control ubiquitin-dependent protein sorting and trafficking.

N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicular bodies and the Golgi, and has a potential role in protein trafficking

N4WBP5A (Ndfip2) belongs to an evolutionarily conserved group of Nedd4-interacting proteins with two homologues in mammalian species. We have previously shown that N4WBP5A expression in Xenopus oocytes results in increased cell-surface expression of the epithelial sodium channel. N4WBPs are characterized by one or two amino terminal PPxY motifs and three transmembrane domains. Here we show that both PPxY motifs of N4WBP5A mediate interaction with WW domains of Nedd4 and that N4WBP5A can physically interact with the WW domains of several Nedd4-family proteins. N4WBP5A is ubiquitinated and ubiquitination does not significantly affect the turnover of N4WBP5A protein. Ubiquitination of N4WBP5A is enhanced by Nedd4 and Nedd4-2 expression. N4WBP5A localizes to the Golgi, vesicles associated with the Golgi complex and to multivesicular bodies. We show that the ectopic expression of N4WBP5A inhibits receptor-mediated endocytosis of labelled epidermal growth factor. N4WBP5A overexpression inhibits accumulation of EGF in large endocytic/lysosomal vesicles suggestive of a role for N4WBP5A in protein trafficking. We propose that N4WBP5A acts as an adaptor to recruit Nedd4 family ubiquitin-protein ligases to the protein trafficking machinery.

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the beta-glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.

A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants

The Green Fluorescent Protein (GFP) from Aequorea victor-in has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER Variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor(TM) Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 +/- 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 mu g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 mu g and 2.11 mu g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfPS-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 mu g mGFP5-ER per mg extractable protein.

Proteomic analysis of normal and malignant prostate tissue to identify novel proteins lost in cancer

BACKGROUND. Alterations of important protein pathways, including loss of prostate secretory granules, and disruption of the prostatic secretory pathway have been identified as early events in malignancy. In this study, proteomics was used to map the differences in protein expression between normal and malignant prostate tissues and to identify and analyze differentially expressed proteins in human prostate tissue with particular regard to the proteins lost in malignancy. METHODS. Small quantities of normal and malignant prostate tissue were taken fresh from 34 radical prostatectomy cases. After histological examination, proteins were solubilized from selected tissues and separated using two-dimensional electrophoresis. Using image analysis, the proteome of normal and malignant tissues were mapped and differentially expressed proteins (present in normal and absent in malignant tissue) were identified and subsequently analyzed using peptide mass finger printing and N-terminal sequencing. Western blotting and immunohistochemistry were performed to examine expression profiles and tissue localization of candidate proteins. RESULTS. Comparison of protein maps of normal and malignant prostate were used to identify 20 proteins which were lost in malignant transformation, including prostate specific antigen (PSA), alpha-l antichymotrypsin (ACT), haptoglobin, and lactoylglutathione lyase. Three of the 20 had not previously been reported in human prostate tissue (Ubiquitin-like NEDD8, calponin, and a follistatin-related protein). Western blotting confirmed differences in the expression profiles of NEDD8 and calponin, and immunohistochemistry demonstrated differences in the cellular localization of these two proteins in normal and malignant prostate glands. CONCLUSIONS. The expression of NEDD8, calponin, and the follistatin-related protein in normal prostate tissues is a novel finding and the role of these important functional proteins in normal prostate and their loss or reduced expression in prostate malignancy warrants further investigations. (C) 2002 Wiley-Liss, Inc.

EDD, the human orthologue of the hyperplastic discs tumour suppressor gene, is amplified and overexpressed in cancer

EDD (E3 isolated by differential display), located at chromosome 8q22.3, is the human orthologue of the Drosophila melanogaster tumour suppressor gene 'hyperplastic discs' and encodes a HECT domain E3 ubiquitin protein-ligase. To investigate the possible involvement of EDD in human cancer, several cancers from diverse tissue sites were analysed for allelic gain or loss (allelic imbalance, AI) at the EDD locus using an EDD-specific microsatellite, CEDD, and other polymorphic microsatellites mapped in the vicinity of the 8q22.3 locus. Of 143 cancers studied, 38 had AI at CEDD (42% of 90 informative cases). In 14 of these cases, discrete regions of imbalance encompassing 8q22.3 were present, while the remainder had more extensive 8q aberrations. AI of CEDD was most frequent in ovarian cancer (22/47 informative cases, 47%), particularly in the serous subtype (16/22, 73%), but was rare in benign and borderline ovarian tumours. AI was also common in breast cancer (31%), hepatocellular carcinoma (46%), squamous cell carcinoma of the tongue (50%) and metastatic melanoma (18%). AI is likely to represent amplification of the EDD gene locus rather than loss of heterozygosity, as quantitative RT-PCR and immunohistochemistry showed that EDD mRNA and protein are frequently overexpressed in breast and ovarian cancers, while among breast cancer cell lines EDD overexpression and increased gene copy number were correlated. These results demonstrate that AI at the EDD locus is common in a diversity of carcinomas and that the EDD gene is frequently overexpressed in breast and ovarian cancer, implying a potential role in cancer progression.

Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+T cell recognition

Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) includes a unique glycine-alanine repeat domain that inhibits the endogenous presentation of cytotoxic T lymphocyte (CTL) epitopes through the class I pathway by blocking proteasome-dependent degradation of this antigen. This immune evasion mechanism has been implicated in the pathogenesis of EBV-associated diseases. Here, we show that cotranslational ubiquitination combined with N-end rule targeting enhances the intracellular degradation of EBNA1, thus resulting in a dramatic reduction in the half-life of the antigen. Using DNA expression vectors encoding different forms of ubiquitinated EBNA1 for in vivo studies revealed that this rapid degradation, remarkably, leads to induction of a very strong CTL response to an EBNA1-specific CTL epitope. Furthermore, this targeting also restored the endogenous processing of HLA class I-restricted CTL epitopes within EBNA1 for immune recognition by human EBV-specific CTLs. These observations provide, for the first time, evidence that the glycine-alanine repeat-mediated proteasomal block on EBNA1 can be reversed by specifically targeting this antigen for rapid degradation resulting in enhanced CD8+ T cell-mediated recognition in vitro and in vivo.

FAM deubiquitylating enzyme is essential for preimplantation mouse embryo development

FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta -catenin and A-F-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure Of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta -catenin such that very little of either protein remained following 72 h culture. The residual beta -catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta -catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta -catenin and AF-6 in vivo. (C) 2001 Elsevier Science Ireland Lid. All rights reserved.

The investigation of optimal bombardment parameters for transient and stable transgene expression in sorghum

This report outlines the development of optimized particle inflow gun (PIG) parameters for producing transgenic sorghum (Sorghum bicolor (L.) Moench). Both transient and stable expression were examined when determining these parameters. The uidA reporter gene (GUS) encoding beta -glucuronidase was used in transient experiments and the green fluorescent protein (GFP) used to monitor stable expression. Initially, optimization was conducted using leaf segments, as the generation of sorghum callus in sufficiently large quantities is time-consuming. Following leaf optimization, experiments were conducted using callus, identifying a high similarity between the two tissue types (r(s) = 0.83). High levels of GUS expression were observed in both leaf and callus material when most distant from the DNA expulsion point, and using a pressure greater than 1800 kPa. A higher level of expression was also observed when the aperture of the helium inlet valve was constricted. Using the optimized conditions (pressure of 2200 kPa, distance to target tissue of 15 cm from the expulsion point, and the aperture of the helium inlet valve at one full turn), three promoters (Ubiquitin, Actin1 and CaMV 35S) were evaluated over a 72-h period using GUS as the reporter gene. A significantly higher number of GUS foci were counted with the Ubiquitin construct over this period, compared to the Actin1 and CaMV 35S constructs. Stable callus sectors (on 2 mg l(-1) bialaphos) with GFP expression were visualized for as long as 6 wk post-bombardment. Using this optimized protocol, several plants were regenerated after having been bombarded with the pAHC20 construct (containing the bar gene), with molecular evidence confirming integration.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the beta -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

The Mallory body as an aggresome: In vitro studies

Prior in vivo studies supported the concept that Mallory bodies (MBs) are aggresomes of cytokeratins 8 and 18. However, to test this hypothesis an in vitro model is needed to study the dynamics of MB formation. Such a study is difficult because MBs have never been induced in tissue culture. Therefore, MBs were first induced in vivo in drug-primed mice and then primary cultures of hepatocytes from these mice were studied. Two approaches were utilized: 1. Primary cultures were transfected with plasmids containing the sequence for cytokeratin 18 (CK 18) tagged with green fluorescent protein (GFP). 2. Immunofluorescent staining was used to localize the ubiquitin-proteasome pathway components involved in MB-aggresome complex formation in primary hepatocyte cultures. The cells were double stained with a ubiquitin antibody and one of the following antibodies: CK 8, CK 18, tubulin, mutant ubiquitin (UBB+ 1), transglutaminase, phosphothreonine, and the 20S and 26S proteasome subunits P25 and Tbp7, respectively. In the first approach, fluorescence was observed in keratin filaments and MBs 48 h after the cells were transfected with the CK 18 GFP plasmid. Nascent cytokeratin 18 was preferentially concentrated in MBs. Less fluorescence was observed in the normal keratin filaments. This indicated that MBs continued to form in vitro. The immunofluorescent staining of the hepatocytes showed that CK 8 and 18, ubiquitin, mutant ubiquitin (UBB+ 1), P25, Tbp7, phosphothreonine, tubulin, and transglutaminase were all located at the border or the interior of the MB. These results support the concept that MBs are aggresomes of CK 8 and CK 18 and are a result of inhibition of the ubiquitin-proteasome pathway of protein degradation possibly caused by UBB+ 1. (C) 2002 Elsevier Science.