Arginine methylation of the RGG box does not appear to regulate ICP27 import during herpes simplex virus infection

Arginine methylation can regulate protein import and export and can modulate protein interactions. Herpes simplex virus 1 (HSV-1) ICP27 is a shuttling protein involved in viral mRNA export. We previously reported that ICP27 is methylated on three arginines within its RGG box and that arginine methylation regulates ICP27 export and its interaction with SRPK1 and Aly/REF. Here, we report that ICP27 was efficiently imported into the nucleus when hypomethylated as determined by Fluorescence Recovery After Photobleaching (FRAP). Furthermore, coimmunoprecipitation of ICP27 with β-importin was not significantly affected by ICP27 hypomethylation. Thus, ICP27 import does not appear to be regulated by arginine methylation.     

Association of ICP0 but not ICP27 with purified virions of herpes simplex virus type 1.

Recent studies have shown that ICP4, one of the major immediate-early proteins of herpes simplex virus type 1 is present within the tegument region of the virion (F. Yao and R. J. Courtney, J. Virol. 63:3338-3344, 1989). With monoclonal antibodies to two additional immediate-early proteins, ICP0 and ICP27, and Western blot (immunoblot) analysis, ICP0, but not ICP27, was also found to be associated with purified virus particles. In an effort to localize the ICP0 within the virion, purified virions were treated with trypsin in the presence and absence of detergent. The data suggest that ICP0 is located within the tegument region of the virion and is not localized in the envelope or within the nucleocapsid. The number of molecules of ICP0 per virion was estimated to be approximately 150.     

The NSR1 gene encodes a protein that specifically binds nuclear localization sequences and has two RNA recognition motifs

We previously identified a protein (p67) in the yeast, Saccharomyces cerevisiae, that specifically recognizes nuclear localization sequences. We report here the partial purification of p67, and the isolation, sequencing, and disruption of the gene (NSR1) encoding this protein. p67 was purified using an affinity column conjugated with a peptide containing the histone H2B nuclear localization sequence from yeast. Using antibodies against p67 we have cloned the gene for this protein. The protein encoded by the NSR1 gene recognizes the wild-type H2B nuclear localization sequence, but does not recognize a mutant H2B sequence that is incompetent for nuclear localization in vivo. Interestingly, the NSR1 protein has two RNA recognition motifs, as well as an acidic NH2 terminus containing a series of serine clusters, and a basic COOH terminus containing arg-gly repeats. We have confirmed the nuclear localization of p67 by immunofluorescence and found that a restricted portion of the nucleus is highlighted. We have also shown that NSR1 (p67) is required for normal cell growth.     

ERp46 binds to AdipoR1, but not AdipoR2, and modulates adiponectin signalling

The pleiotropic effects of the insulin-sensitizing adipokine adiponectin are mediated, at least in part, by two seven-transmembrane domain receptors AdipoR1 and AdipoR2. Recent reports indicate a role for AdipoR-binding proteins, namely APPL1, RACK1 and CK2β, in proximal signal transduction events. Here we demonstrate that endoplasmic reticulum protein 46 (ERp46) interacts specifically with AdipoR1 and provide evidence that ERp46 modulates adiponectin signalling. Co-immunoprecipitation followed by mass spectrometry identified ERp46 as an AdipoR1-, but not AdipoR2-, interacting protein. Analysis of truncated constructs and GST-fusion proteins revealed the interaction was mediated by the cytoplasmic, N-terminal residues (1-70) of AdipoR1. Indirect immunofluorescence microscopy and subcellular fractionation studies demonstrated that ERp46 was present in the ER and the plasma membrane (PM). Transient knockdown of ERp46 increased the levels of AdipoR1, and AdipoR2, at the PM and this correlated with increased adiponectin-stimulated phosphorylation of AMPK. In contrast, adiponectin-stimulated phosphorylation of p38MAPK was reduced following ERp46 knockdown. Collectively these results establish ERp46 as the first AdipoR1-specific interacting protein and suggest a role for ERp46 in adiponectin receptor biology and adiponectin signalling.     

Rotavirus VP1 alone specifically binds to the 3' end of viral mRNA, but the interaction is not sufficient to initiate minus-strand synthesis.

Recent studies have shown that disrupted (open) rotavirus cores have an associated replicase activity which supports the synthesis of dsRNA from viral mRNA in a cell-free system (D. Chen, C. Q.-Y. Zeng, M. J. Wentz, M. Gorziglia, M. K. Estes, and R. F. Ramig, J. Virol. 68:7030-7039, 1994). To determine which of the core proteins, VP1, VP2, or VP3, recognizes the template mRNA during RNA replication, SA11 open cores were incubated with 32P-labeled RNA probes of viral and nonviral origin and the reaction mixtures were analyzed for the formation of RNA-protein complexes by gel mobility shift assay. In mixtures containing a probe representing the 3' end of SA11 gene 8 mRNA, two closely migrating RNA-protein complexes, designated s and f, were detected. The interaction between the RNA and protein of the s and f complexes was shown to be specific by competitive binding assay with tRNA and brome mosaic virus RNA. By electrophoretic analysis of RNA-protein complexes recovered from gels, VP1 was shown to be the only viral protein component of the complexes, thereby indicating that VP1 specifically recognizes the 3' end of gene 8 mRNA. Analysis of VP1 purified from open cores by glycerol gradient centrifugation verified that VP1 recognizes the 3' end of viral mRNA but also showed that in the absence of other viral proteins, VP1 lacks replicase activity. When reconstituted with VP2-rich portions of the gradient, VP1 stimulated levels of replicase activity severalfold. These data indicate that VP1 can bind to viral mRNA in the absence of any other viral proteins and suggest that VP2 must interact with the RNA-protein complex before VP1 gains replicase activity.     

Full-length RAG-2, and not full-length RAG-1, specifically suppresses RAG-mediated transposition but not hybrid joint formation or disintegration

RAG-1 and RAG-2 initiate V(D)J recombination by introducing DNA breaks at recombination signal sequences flanking a pair of antigen receptor gene segments. Occasionally, the RAG proteins mediate two other alternative DNA rearrangements in vivo: the rejoining of signal and coding ends and the transposition of signal ends into unrelated DNA. In contrast, truncated, catalytically active "core" RAG proteins readily catalyze these reactions in vitro, suggesting that full-length RAG proteins directly or indirectly suppress these undesired reactions in vivo. To discriminate between direct and indirect suppression models, full-length RAG proteins were purified and characterized in vitro. From mammalian cells, full-length RAG-1 is readily purified with core RAG-2 but not full-length RAG-2 and vice versa. Despite differences in DNA binding activity, recombinase containing either core or full-length RAG-1 or RAG-2 possess comparable cleavage, rejoining, and end-processing activity, as well as similar usage preferences for canonical versus cryptic recombination signals. However, recombinase containing full-length RAG-2, but not full-length RAG-1, exhibits dramatically reduced transposition activity in vitro. These data suggest RAG-mediated transposition and rejoining are differentially regulated by the full-length RAG proteins in vivo (the former directly by RAG-2 and the latter indirectly through other factors) and argue that noncore portions of the RAG proteins have little or no direct influence over V(D)J recombinase site specificity.     

The cytoplasmic region of mouse Fc gamma RIIb1, but not Fc gamma RIIb2, binds phospholipid membranes

The cytoplasmic regions of the mouse low-affinity Fc gamma RII isoforms, Fc gamma RIIb1 and Fc gamma RIIb2, play key roles in signal transduction by mediating different cellular functions. The Fc gamma RIIb1 (94 residues) and Fc gamma RIIb2 (47 residues) cytoplasmic regions are generated by differential mRNA splicing in which a single aspartic acid residue in Fc gamma RIIb2 is replaced by a 48-residue insert in Fc gamma RIIb1. In previous work, quantities of the mFc gamma RIIb1 and mFc gamma RIIb2 cytoplasmic regions were generated, and their secondary structures were examined in different solutions with circular dichroism [Chen, L., Thompson, N. L., and Pielak, G. J. (1997) Protein Sci. 6, 1038-1046]. In the work described here, steady-state light scattering was used to investigate possible interactions of the two isolated cytoplasmic regions with phospholipid vesicles. Three phospholipid compositions were examined: phosphatidylserine/phosphatidylcholine (PS/PC) (25/75, mol/mol); phosphatidylinositol bisphosphate/phosphatidylcholine (PIP2/PC) (25/75, mol/mol); and pure phosphatidylcholine (PC). Binding was examined in the presence and absence of Ca2+. The mFc gamma RIIb1 cytoplasmic peptide binds PS/PC vesicles weakly in the absence of Ca2+ and more strongly in the presence of Ca2+. For PIP2/PC vesicles, the behavior is reversed; binding is weak in the presence of Ca2+ and stronger in its absence. The mFc gamma RIIb1 peptide also weakly binds pure PC vesicles, in a Ca2+-independent manner. The mFc gamma RIIb2 cytoplasmic peptide does not bind, in the presence or absence of Ca2+, to PS/PC, PIP2/PC, or PC vesicles. The implications of these results for the mechanisms of signal transduction mediated by the two mFc gamma RII cytoplasmic regions are discussed.     

The membrane-bound DnaJ protein located at the cytosolic site of glyoxysomes specifically binds the cytosolic isoform 1 of Hsp70 but not other Hsp70 species.

DnaJ proteins are located in various compartments of the eukaryotic cell. As previously shown, peroxisomes and glyoxysomes possess a membrane-anchored form of DnaJ protein located on the cytosolic face. Hints as to how the membrane-bound co-chaperone interacts with cytosolic soluble chaperones were obtained by examining the affinity between the DnaJ protein and various potential partners of the Hsp70 family. Two genes encoding cytosolic Hsp70 isoforms were isolated and characterized from cucumber cotyledons. In addition, cDNAs encoding Hsp70 forms attributed to the cytosol, plastids and the lumen of the endoplasmic reticulum were prepared. His-tagged DnaJ proteins and glutathione S-transferase-Hsp70 fusion proteins were constructed. Using these tools, it was demonstrated that the soluble His-tagged form of DnaJ protein exclusively binds the cytosolic isoform 1 of Hsp70. This interaction was further analyzed by characterizing the interaction between the glyoxysome-bound form of the DnaJ protein and various isoforms of Hsp70. Specific binding to the glyoxysomal surface was only observed in the case of cytosolic isoform 1 of Hsp70. This interaction was strictly dependent on the presence of ADP. Glyoxysomes did not bind other cytosolic or plastidic isoforms or the BiP-related form of Hsp70. Analyzing the enzymatic properties of cytosolic Hsp70s, we showed that the ATPase-modulating activity of DnaJ was highest when isoform 1 was assayed. Collectively, the data indicate that the partner of the DnaJ protein anchored at the glyoxysomal membrane is the cytosolic isoform 1 of Hsp70. In addition to the chaperones located at the surface of glyoxysomes, two isoforms of Hsp70 and one soluble form of DnaJ protein were detected in the glyoxysomal matrix.     

The R6A-1 peptide binds to switch II of Galphai1 but is not a GDP-dissociation inhibitor

Heterotrimeric G-proteins are molecular switches that convert signals from membrane receptors into changes in intracellular physiology. Recently, several peptides that bind heterotrimeric G-protein alpha subunits have been isolated including the novel Galpha(i1).GDP binding peptides R6A and KB-752. The R6A peptide and its minimized derivative R6A-1 interact with Galpha(i1).GDP. Based on spectroscopic analysis of BODIPYFL-GTPgammaS binding to Galpha(i1), it has been reported that R6A-1 has guanine nucleotide dissociation inhibitor (GDI) activity against Galpha(i1) [W.W. Ja, R.W. Roberts, Biochemistry 43 (28) (2004) 9265-9275]. Using radioligand binding, we show that R6A-1 is not a GDI for Galpha(i1) subunits. Furthermore, we demonstrate that R6A-1 reduces the fluorescence quantum yield of the Galpha(i1)-BODIPYFL-GTPgammaS complex, thus explaining the previously reported GDI activity as a fluorescence artifact. We further show that R6A-1 has significant sequence similarity to the guanine nucleotide exchange factor peptide KB-752 that binds to switch II of Galpha(i1). We use competitive binding analysis to show that R6A-1 also binds to switch II of Galpha subunits.     

Simian virus 40 (SV40) T antigen binds specifically to double-stranded DNA but not to single-stranded DNA or DNA/RNA hybrids containing the SV40 regulatory sequences.

Simian virus 40 T antigen has been shown previously to bind specifically with high affinity to sites within the regulatory region of double-stranded simian virus 40 DNA. Using competition filter binding and the DNA-binding immunoassay, we show that T antigen did not bind specifically to either early or late single-stranded DNA containing these binding sites. Moreover, T antigen did not bind these sequences present in single-stranded RNA, RNA/RNA duplexes, or RNA/DNA hybrids. T antigen did, however, bind as efficiently to single-stranded DNA-cellulose as to double-stranded DNA-cellulose. This binding was nonspecific because it was independent of the presence of T-antigen-binding sites. The implications of these observations are discussed.     

p18Ink4c, but not p27Kip1, collaborates with Men1 to suppress neuroendocrine organ tumors

Mutant mice lacking both cyclin-dependent kinase (CDK) inhibitors p18(Ink4c) and p27(Kip1) develop a tumor spectrum reminiscent of human multiple endocrine neoplasia (MEN) syndromes. To determine how p18 and p27 genetically interact with Men1, the tumor suppressor gene mutated in familial MEN1, we characterized p18-Men1 and p27-Men1 double mutant mice. Compared with their corresponding single mutant littermates, the p18(-/-); Men1(+/-) mice develop tumors at an accelerated rate and with an increased incidence in the pituitary, thyroid, parathyroid, and pancreas. In the pituitary and pancreatic islets, phosphorylation of the retinoblastoma (Rb) protein at both CDK2 and CDK4/6 sites was increased in p18(-/-) and Men1(+/-) cells and was further increased in p18(-/-); Men1(+/-) cells. The remaining wild-type Men1 allele was lost in most tumors from Men1(+/-) mice but was retained in most tumors from p18(-/-); Men1(+/-) mice. Combined mutations of p27(-/-) and Men1(+/-), in contrast, did not exhibit noticeable synergistic stimulation of Rb kinase activity, cell proliferation, and tumor growth. These results demonstrate that functional collaboration exists between p18 and Men1 and suggest that Men1 may regulate additional factor(s) that interact with p18 and p27 differently.     

H3K27me1 is E(z) in animals,but not in plants

Post-translational modifications of histones play key roles in the regulation of gene expression and chromatin structure in eukaryotes. Methylation of histone 3 on lysine 27 (H3K27) is one of the most common and well-studied histone post-translational modifications. The vast majority of research on this histone residue, however, has focused on the trimethylated form (H3K27me3). Despite occurring at higher levels than H3K27me3 in animals and plants, the monomethylated form of H3K27 (H3K27me1) remains relatively poorly characterized. The absence of information concerning H3K27me1 is due in large part to the fact that the enzymes catalyzing this epigenetic mark were only recently identified. In this article, we highlight new findings concerning H3K27me1, including the identification of two plant-specific H3K27 monomethyltransferases that are required for gene silencing and heterochromatin condensation. We also discuss the emerging similarities and differences in H3K27 methylation in plant and animal systems.