Protein Arginine Methyltransferase Expression Affects Ectomycorrhizal Symbiosis and the Regulation of Hormone Signaling Pathways.

The genomes of all eukaryotic organisms, from small unicellular yeasts to humans, include members of the protein arginine methyltransferase (PRMT) family. These enzymes affect gene transcription, cellular signaling, and function through the posttranslational methylation of arginine residues. Mis-regulation of PRMTs results in serious developmental defects, disease, or death, illustrating the importance of these enzymes to cellular processes. Plant genomes encode almost the full complement of PRMTs found in other higher organisms, plus an additional PRMT found uniquely in plants, PRMT10. Here, we investigate the role of these highly conserved PRMTs in a process that is unique to perennial plants-the development of symbiosis with ectomycorrhizal fungi. We show that PRMT expression and arginine methylation is altered in the roots of the model tree Eucalyptus grandis by the presence of its ectomycorrhizal fungal symbiont Pisolithus albus. Further, using transgenic modifications, we demonstrate that E. grandis-encoded PRMT1 and PRMT10 have important but opposing effects in promoting this symbiosis. In particular, the plant-specific EgPRMT10 has a potential role in the expression of plant hormone pathways during the colonization process and its overexpression reduces fungal colonization success.

Protein arginine methylation: an emerging regulator of the cell cycle.

Protein arginine methylation is a common post-translational modification where a methyl group is added onto arginine residues of a protein to alter detection by its binding partners or regulate its activity. It is known to be involved in many biological processes, such as regulation of signal transduction, transcription, facilitation of protein-protein interactions, RNA splicing and transport. The enzymes responsible for arginine methylation, protein arginine methyltransferases (PRMTs), have been shown to methylate or associate with important regulatory proteins of the cell cycle and DNA damage repair pathways, such as cyclin D1, p53, p21 and the retinoblastoma protein. Overexpression of PRMTs resulting in aberrant methylation patterns in cancers often correlates with poor recovery prognosis. This indicates that protein arginine methylation is also an important regulator of the cell cycle, and consequently a target for cancer regulation. The effect of protein arginine methylation on the cell cycle and how this emerging key player of cell cycle regulation may be used in therapeutic strategies for cancer are the focus of this review.

Root morphogenic pathways in Eucalyptus grandis are modified by the activity of protein arginine methyltransferases.

BACKGROUND: Methylation of proteins at arginine residues, catalysed by members of the protein arginine methyltransferase (PRMT) family, is crucial for the regulation of gene transcription and for protein function in eukaryotic organisms. Inhibition of the activity of PRMTs in annual model plants has demonstrated wide-ranging involvement of PRMTs in key plant developmental processes, however, PRMTs have not been characterised or studied in long-lived tree species. RESULTS: Taking advantage of the recently available genome for Eucalyptus grandis, we demonstrate that most of the major plant PRMTs are conserved in E. grandis as compared to annual plants and that they are expressed in all major plant tissues. Proteomic and transcriptomic analysis in roots suggest that the PRMTs of E. grandis control a number of regulatory proteins and genes related to signalling during cellular/root growth and morphogenesis. We demonstrate here, using chemical inhibition of methylation and transgenic approaches, that plant type I PRMTs are necessary for normal root growth and branching in E. grandis. We further show that EgPRMT1 has a key role in root hair initiation and elongation and is involved in the methylation of ß-tubulin, a key protein in cytoskeleton formation. CONCLUSIONS: Together, our data demonstrate that PRMTs encoded by E. grandis methylate a number of key proteins and alter the transcription of a variety of genes involved in developmental processes. Appropriate levels of expression of type I PRMTs are necessary for the proper growth and development of E. grandis roots.

Proenergetic effects of resveratrol in the murine neuronal cell line Neuro2a.

SCOPE: Energy deficit is a common characteristic of neurodegenerative disorders, including Alzheimer's disease. Adenosine monophosphate activated protein kinase (AMPK) is a key enzyme maintaining energy balance by regulating the cellular uptake of glucose, ß-oxidation of fatty acids, and expression of glucose transporter 4. Since resveratrol has been shown to increase the activity of AMPK, we hypothesized that it might influence energy metabolism in a model neuron-like cell line, murine Neuro2a cells. METHODS AND RESULTS: Resveratrol caused an elevation of adenosine triphosphate (ATP) and guanosine triphosphate (GTP) in a dose-dependent manner. The highest ATP and GTP levels achieved by treatment with resveratrol were 70.3 ± 8.2 nmol/mg protein (1.9-fold of control) and 27.2 ± 4.0 nmol/mg protein (1.7-fold of control), respectively, when cells were treated with 100 µM resveratrol for 6 h. Interestingly, increases in the total sum of all adenine nucleotides were found upon addition of resveratrol. Despite these increases in ATP, GTP, and the total adenine nucleotide pool, resveratrol treatment led to a decrease in glucose consumption and lactate release, suggesting that resveratrol does not increase energy production (e.g. via AMPK kinase activation) but rather inhibits energy-consuming processes. CONCLUSION: Resveratrol increases the levels of ATP and GTP, but without creating an additional glucose demand.

Laser-activated adhesive films for sutureless median nerve anastomosis.

A novel chitosan adhesive film that incorporates the dye 'Rose Bengal' (RB) was used in conjunction with a green laser to repair transected rat median nerves in vivo. Histology and electrophysiological recording assessed the impact of the laser-adhesive technique on nerves. One week post-operatively, the sham-control group (laser-adhesive technique applied on un-transected nerves) conserved the average number and size of myelinated fibres in comparison to its contralateral side and electrophysiological recordings demonstrated no significant difference with un-operated nerves. Twelve weeks after the laser-adhesive anastomoses, nerves were in continuity with regenerated axons that crossed the anastomotic site.

In vitro cell compatibility study of rose bengal-chitosan adhesives.

BACKGROUND AND OBJECTIVES: Photochemical tissue bonding (PTB) using rose bengal (RB) in conjunction with light is an alternative technique to repair tissue without suturing. It was recently demonstrated that laser-irradiated chitosan films, incorporating RB, bonded firmly to calf intestine in vitro. It is thus required to investigate the possible cytotoxic effects of the RB-chitosan adhesive on cells before testing its application to in vivo models. MATERIALS AND METHODS: Adhesive films, based on chitosan and containing ~0.1 wt% RB were fabricated. Their cytotoxicity was assessed by growing human and murine fibroblasts either in media in which adhesive strips had been incubated, or directly on the adhesive. The adhesive was either laser-irradiated or not. Cells were stained after 48 hours with Trypan blue and the number of live and dead cells was recorded for cell viability. RESULTS: Murine and human fibroblasts grew confluent on the adhesives with no apparent morphological changes or any exclusion zone. Cell numbers of murine fibroblasts were not significantly different when cultured in media that was extracted from irradiated (86 ± 7%) and non-irradiated adhesive (89 ± 4%). A similar result was obtained for the human fibroblasts. CONCLUSIONS: These findings support that the RB-chitosan films induced negligible toxicity and growth retardation in murine and human fibroblasts.

Photochemical tissue bonding with chitosan adhesive films.

BACKGROUND: Photochemical tissue bonding (PTB) is a promising sutureless technique for tissue repair. PTB is often achieved by applying a solution of rose bengal (RB) between two tissue edges, which are irradiated by a green laser to crosslink collagen fibers with minimal heat production. In this study, RB has been incorporated in chitosan films to create a novel tissue adhesive that is laser-activated. METHODS: Adhesive films, based on chitosan and containing ~0.1 wt% RB were manufactured and bonded to calf intestine by a solid state laser (λ = 532 nm, Fluence~110 J/cm2, spot size~0.5 cm). A single-column tensiometer, interfaced with a personal computer, tested the bonding strength. K-type thermocouples recorded the temperature (T) at the adhesive-tissue interface during laser irradiation. Human fibroblasts were also seeded on the adhesive and cultured for 48 hours to assess cell growth. RESULTS: The RB-chitosan adhesive bonded firmly to the intestine with adhesion strength of 15 ± 2 kPa, (n = 31). The adhesion strength dropped to 0.5 ± 0.1 (n = 8) kPa when the laser was not applied to the adhesive. The average temperature of the adhesive increased from 26°C to 32°C during laser exposure. Fibroblasts grew confluent on the adhesive without morphological changes. CONCLUSION: A new biocompatible chitosan adhesive has been developed that bonds photochemically to tissue with minimal temperature increase.

Proteolytic cleavage of HIV-1 GFP-Vpr fusions at novel sites within virions and living cells: concerns for intracellular trafficking studies.

Fluorescent labelling of the highly conserved HIV-1 accessory protein Vpr (Viral Protein R) with GFP or variants thereof has proved a valuable approach to track Vpr and/or HIV-1 subcellular localisation in vivo. Our analysis in transfected mammalian cells expressing GFP-Vpr fusion protein, as well as within virus derived there from, documents site-specific proteolytic cleavage of the GFP-Vpr fusion protein. Western analysis revealed that transfected mammalian cells harbour a C-terminally truncated variant of Vpr in addition to full-length GFP-Vpr. Further, virions derived from these GFP-Vpr expressing cells show protein in which the GFP-tag has been additionally cleaved from the Vpr protein. Endogenous HIV protease (PR) activity was shown to be responsible for the latter, as addition of Saquinavir, a potent PR inhibitor abolished the cleavage. Since many previous studies have relied on imaging the GFP fluorescence of GFP-Vpr, it would appear that the results may not reflect intact GFP-Vpr.

Impaired nuclear import and viral incorporation of Vpr derived from a HIV long-term non-progressor.

We previously reported an epidemiologically linked HIV-1 infected patient cohort in which a long-term non-progressor (LTNP) infected two recipients who then exhibited normal disease progression. Expression of patient-derived vpr sequences from each of the three cohort members in mammalian cells tagged with GFP revealed a significant reduction in Vpr nuclear import and virion incorporation uniquely from the LTNP, whereas Vpr from the two progressing recipients displayed normal localisation and virion incorporation, implying a link between efficient Vpr nuclear import and HIV disease progression. Importantly, an F72L point mutation in the LTNP was identified for the first time as being uniquely responsible for decreased Vpr nuclear import.

The N-terminal basic domain of the HIV-1 matrix protein does not contain a conventional nuclear localization sequence but is required for DNA binding and protein self-association.

The HIV p17 or matrix (MA) protein has long been implicated in the process of nuclear import of the HIV genome and thus the ability of the virus to infect nondividing cells such as macrophages. While it has been demonstrated that MA is not absolutely required for this process, debate continues to surround the subcellular targeting properties of MA and its potential contribution to nuclear import of the HIV cDNA. Through the use of in vitro techniques we have determined that, despite the ability of MA to interact with importins, the full-length protein fails to enter the nucleus of cells. While MA does contain a region of basic amino acids within its N-terminus which can confer nuclear accumulation of a fusion protein, we show that this is due to nuclear retention mediated by DNA binding and does not represent facilitated import. Importantly, we show that the 26KK residues of MA, previously thought to be part of a nuclear localization sequence, are absolutely required for a number of MA's functions including its ability to bind DNA and RNA and its propensity to form high-order multimers/protein aggregates. The results presented here indicate that the N-terminal basic domain of MA does not appear likely to play a role in HIV cDNA nuclear import; rather this region appears to be a crucial structural and functional motif whose integrity is required for a number of other roles performed by MA during viral infection.

The biarsenical dye Lumio exhibits a reduced ability to specifically detect tetracysteine-containing proteins within live cells.

Investigating the localisation of proteins within live cells via fluorescence microscopy typically involves the fusion of the protein of interest to a large fluorescent protein such as green fluorescent protein (GFP). Alternate fluorescent labelling technologies such as the fluorescent biarsenical dye molecules (e.g. FlAsH, ReAsH) are preferable to the use of large fusion proteins in many respects and allow greater flexibility in terms of the location of the labelling site. We assessed the ability of the FlAsH-derived biarsenical dye molecule Lumio to label a range of tetracysteine containing proteins within live cells and report that although in some circumstances Lumio is capable of positively detecting such proteins, the sensitivity and specificity of labelling is significantly reduced, making the Lumio-labelling system unsuitable for the detection of a wide range of protein within live cells.

Protein methylation is required to maintain optimal HIV-1 infectivity.

BACKGROUND: Protein methylation is recognized as a major protein modification pathway regulating diverse cellular events such as protein trafficking, transcription, and signal transduction. More recently, protein arginine methyltransferase activity has been shown to regulate HIV-1 transcription via Tat. In this study, adenosine periodate (AdOx) was used to globally inhibit protein methyltransferase activity so that the effect of protein methylation on HIV-1 infectivity could be assessed. RESULTS: Two cell culture models were used: HIV-1-infected CEM T-cells and HEK293T cells transfected with a proviral DNA plasmid. In both models, AdOx treatment of cells increased the levels of virion in culture supernatant. However, these viruses had increased levels of unprocessed or partially processed Gag-Pol, significantly increased diameter, and displayed reduced infectivity in a MAGI X4 assay. AdOx reduced infectivity equally in both dividing and non-dividing cells. However, infectivity was further reduced if Vpr was deleted suggesting virion proteins, other than Vpr, were affected by protein methylation. Endogenous reverse transcription was not inhibited in AdOx-treated HIV-1, and infectivity could be restored by pseudotyping HIV with VSV-G envelope protein. These experiments suggest that AdOx affects an early event between receptor binding and uncoating, but not reverse transcription. CONCLUSION: Overall, we have shown for the first time that protein methylation contributes towards maximal virus infectivity. Furthermore, our results also indicate that protein methylation regulates HIV-1 infectivity in a complex manner most likely involving the methylation of multiple viral or cellular proteins and/or multiple steps of replication.

Point mutations in the C-terminus of HIV-1 gp160 reduce apoptosis and calmodulin binding without affecting viral replication.

One hallmark of AIDS progression is a decline in CD4+ T lymphocytes, though the mechanism is poorly defined. There is ample evidence that increased apoptosis is responsible for some, if not all, of the decline. Prior studies have shown that binding of cellular calmodulin to the envelope glycoprotein (Env) of HIV-1 increases sensitivity to fas-mediated apoptosis and that calmodulin antagonists can block this effect. We show that individual mutation of five residues in the C-terminal calmodulin-binding domain of Env is sufficient to significantly reduce fas-mediated apoptosis in transfected cells. The A835W mutation in the cytoplasmic domain of gp41 eliminated co-immunoprecipitation of Env with calmodulin in studies with stably transfected cells. Four point mutations (A835W, A838W, A838I, and I842R) and the corresponding region of HIV-1 HXB2 were cloned into the HIV-1 proviral vector pNL4-3 with no significant effect on viral production or envelope expression, although co-immunoprecipitation of calmodulin and Env was decreased in three of these mutant viruses. Only wild-type envelope-containing virus induced significantly elevated levels of spontaneous apoptosis by day 5 post-infection. Fas-mediated apoptosis levels positively correlated with the degree of calmodulin co-immunoprecipitation, with the lowest apoptosis levels occurring in cells infected with the A835W envelope mutation. While spontaneous apoptosis appears to be at least partially calmodulin-independent, the effects of HIV-1 Env on fas-mediated apoptosis are directly related to calmodulin binding.

Evidence for host-driven selection of the HIV type 1 vpr gene in vivo during HIV disease progression in a transfusion-acquired cohort.

An epidemiologically linked HIV-1-infected cohort, in which a nonprogressor donor infected two recipients who progressed to AIDS, was examined. Sequence analysis, over time, of HIV-1 vpr gene quasispecies from uncultured peripheral blood cells revealed an insertion of arginine at position 90 altering a highly conserved C-terminal motif, believed to play a role in Vpr nuclear targeting. Full genome analysis from each patient showed no gene defects in other gene regions, implying that the mutational selection was unique to the vpr gene. A detailed analysis of the vpr quasispecies showed very little amino acid diversity in the nonprogressing donor, whereas, following viral transmission, the amino acid diversity increased dramatically over time in tandem with disease progression in the two recipients. Although the R insertion at position 90 was present in all three individuals, the variable degree of additional amino acid changes over time may have influenced HIV disease in the nonprogressor donor and the two progressing recipients. These data provide the first evidence in favor of vpr gene evolution over time, which was host-driven. The status of the nonprogressing donor was consistent with a highly protective B-57 HLA type, which was absent in the two progressing recipients, implying a role for host HLA type and other immunologic selective pressures in vpr gene selection in vivo.

A 100-amino acid truncation in the cytoplasmic tail of glycoprotein 41 in the reference HIV type 1 strain RF.

Truncations of the cytoplasmic tail of the HIV-1 transmembrane (TM) protein are rare and almost always markedly reduce virus infectivity. We describe a truncation of the gp41 cytoplasmic tail in the commonly used early HIV-1 reference strain RF. This truncation apparently arose after continuous passage in H9 cells. We detected the truncation by Western blot as a size decrease in RF gp41 from 46 to approximately 34 kDa. The reduced size of RF gp41 observed was not due to differences in glycosylation. Viral DNA sequencing confirmed that a point mutation at Env residue 740 (Trp) introduced a premature stop codon, resulting in a 100-amino acid (13-kDa) truncation of the gp41 C terminus. This truncated RF species, termed RF(gp34), was characterized phenotypically by growth in Hut78 cells. Compared with other B clade HIV strains (IIIB, SF2, and NL4.3), RF(gp34) induced massive syncytia. Importantly, RF(gp34) also productively infected peripheral blood mononuclear cells in vitro.