SV40 large T antigen is modified with O-linked N-acetylglucosamine but not with other forms of glycosylation

SV40 large T antigen has been reported to be modified with several different sugars including N-acetylglucosamine, galactose, and mannose. In this report we have reexamined the glycosylation of T antigen and found that while we could detect modification with N-acetylglucosamine, we could not detect any other sugars on the protein. Surprisingly, even though [3H]galactose could be metabolically incorporated into the protein, analysis showed that all of the radioactivity in T antigen had been converted to other species. The N-acetylglucosamine was demonstrated to be linked to the protein in the form of O-linked N- acetylglucosamine, the best characterized form of nuclear and cytoplasmic glycosylation in mammalian systems. We have localized the major site of glycosylation to the amino terminal portion of the molecule. Analysis of mutated T antigen where serines 111/112 were substituted with alanine suggest that these residues constitute a glycosylation site on the protein. These two serines fall within a typical O-linked N-acetylglucosamine glycosylation site (PSS) and are also known to be phosphorylated. Thus, it is likely that competition between phosphorylation and glycosylation occurs at this site.      

Muscarinic receptor stimulation increases regulators of G-protein signaling 2 mRNA levels through a protein kinase C-dependent mechanism.

RGS2, a member of the Regulators of G-protein Signaling family, modulates the activity of G-proteins coupled to the phosphoinositide signal transduction system, but little is known about what regulates RGS2. In human neuroblastoma SH-SY5Y cells stimulation of muscarinic receptors by carbachol activates phosphoinositide signaling and also caused a rapid, large, and long lasting increase in RGS2 mRNA levels. Direct activation of protein kinase C also rapidly increased RGS2 mRNA levels. Inhibition of protein kinase C with Ro31-8220, GF109203x, or Go6976 or down-regulation of protein kinase C inhibited increases in RGS2 mRNA levels induced by carbachol or by the activation of protein kinase C. Blockade of calcium signaling did not alter carbachol-induced increases in RGS2 mRNA levels. Neither activation of epidermal growth factor receptors nor stimulation of cyclic AMP production with forskolin increased RGS2 mRNA levels. Pretreatment with actinomycin D blocked increases in RGS2 mRNA levels but caused a surprisingly small, although statistically significant, partial blockade of protein kinase C-mediated feedback inhibition of carbachol-induced phosphoinositide hydrolysis. Thus, RGS2 mRNA levels are increased by activation of muscarinic receptors coupled to the phosphoinositide signal transduction system through a protein kinase C-dependent mechanism. This action may contribute to negative feedback control of this signaling cascade, but because the small contribution to negative feedback contrasts with the large and prolonged elevations in RGS2 mRNA levels, we speculate that its primary role may be in modulating other signaling components.     

Self-association of the Lentivirus protein, Nef

Background

The HIV-1 pathogenic factor, Nef, is a multifunctional protein present in the cytosol and on membranes of infected cells. It has been proposed that a spatial and temporal regulation of the conformation of Nef sequentially matches Nef's multiple functions to the process of virion production. Further, it has been suggested that dimerization is required for multiple Nef activities. A dimerization interface has been proposed based on intermolecular contacts between Nefs within hexagonal Nef/FynSH3 crystals. The proposed dimerization interface consists of the hydrophobic B-helix and flanking salt bridges between R105 and D123. Here, we test whether Nef self-association is mediated by this interface and address the overall significance of oligomerization.

Results

By co-immunoprecipitation assays, we demonstrated that HIV-1Nef exists as monomers and oligomers with about half of the Nef protomers oligomerized. Nef oligomers were found to be present in the cytosol and on membranes. Removal of the myristate did not enhance the oligomerization of soluble Nef. Also, SIVNef oligomerizes despite lacking a dimerization interface functionally homologous to that proposed for HIV-1Nef. Moreover, HIV-1Nef and SIVNef form hetero-oligomers demonstrating the existence of homologous oligomerization interfaces that are distinct from that previously proposed (R105-D123). Intracellular cross-linking by formaldehyde confirmed that SF2Nef dimers are present in intact cells, but surprisingly self-association was dependent on R105, but not D123. SIV_MAC239Nef can be cross-linked at its only cysteine, C55, and SF2Nef is also cross-linked, but at C206 instead of C55, suggesting that Nefs exhibit multiple dimeric structures. ClusPro dimerization analysis of HIV-1Nef homodimers and HIV-1Nef/SIVNef heterodimers identified a new potential dimerization interface, including a dibasic motif at R105-R106 and a six amino acid hydrophobic surface.

Conclusions

We have demonstrated significant levels of intracellular Nef oligomers by immunoprecipitation from cellular extracts. However, our results are contrary to the identification of salt bridges between R105 and D123 as necessary for self-association. Importantly, binding between HIV-1Nef and SIVNef demonstrates evolutionary conservation and therefore significant function(s) for oligomerization. Based on modeling studies of Nef self-association, we propose a new dimerization interface. Finally, our findings support a stochastic model of Nef function with a dispersed intracellular distribution of Nef oligomers.     

Identification of an antiapoptotic protein complex at death receptors

Stimulation of death receptors activates the extrinsic apoptotic signaling pathway that leads to cell death. Although many steps of this apoptotic signaling cascade are known, few mechanisms that counterbalance the death signal have been described. We identified an antiapoptotic protein complex associated with death receptors that contains glycogen synthase kinase-3 (GSK3), DDX3 and cellular inhibitor of apoptosis protein-1 (cIAP-1). GSK3, DDX3 and cIAP-1 are associated in cells with each other and with death receptors. Blocking the actions of GSK3 or DDX3 potentiated caspase-3 activation induced by stimulation of four different death receptors in several types of cells. GSK3 restrained apoptotic signaling by inhibiting formation of the death-inducing signaling complex and caspase-8 activation. Stimulated death receptors surmount the antiapoptotic complex by causing GSK3 inactivation and cleavage of DDX3 and cIAP-1 to enable progression of the apoptotic signaling cascade, but the antiapoptotic complex remains functional in cancer cells resistant to death receptor stimulation, a resistance that is overcome by GSK3 inhibitors. Thus, an antiapoptotic complex of GSK3, DDX3 and cIAP-1 caps death receptors, providing a checkpoint to counterbalance apoptotic signaling.     

Chronic Lithium Treatment Impairs Phosphatidylinositol Hydrolysis in Membranes from Rat Brain Regions

Membranes prepared from rat brain regions were used to measure the receptor-coupled and/or guanine nucleotide-binding protein (G protein)-mediated hydrolysis of exogenous [3H]phosphatidylinositol ([3H]PI). Guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and NaF (in the presence of AlCl3) caused concentration-dependent stimulations of [3H]PI hydrolysis, supporting the conclusion that G proteins mediating [3H]PI hydrolysis can be activated in this preparation. Neither of these responses was altered by in vitro incubation with 8 mM LiCl, but both were reduced in hippocampal, striatal, and cortical membranes from rats that had been treated with lithium for 4 weeks compared with controls. Two cholinergic agonists, carbachol and pilocarpine, induced no hydrolysis of [3H]PI unless GTP gamma S was also present, in which case each equally stimulated [3H]PI hydrolysis above that obtained with GTP gamma S alone. In the presence of GTP gamma S several excitatory amino acid agonists stimulated [3H]PI hydrolysis to an extent similar to that of carbachol. After chronic lithium treatment, [3H]PI hydrolysis stimulated by carbachol was significantly attenuated, but the response to quisqualate was unaffected. Therefore, lithium added in vitro does not have an effect on cholinergic receptor- or G protein-mediated [3H]PI hydrolysis, but each of these is reduced by chronic lithium treatment. Because exogenous [3H]PI was provided as the substrate, it is evident that the inhibitory effect of chronic lithium treatment cannot be due to substrate depletion. Impaired function of G proteins appears to be the most likely mechanism accounting for attenuated [3H]PI hydrolysis after chronic administration of lithium.     

REGULATION OF ACETYLCHOLINE SYNTHESIS: CONTROL OF CHOLINE TRANSPORT AND ACETYLATION IN SYNAPTOSOMES

Abstract— The high affinity transport of choline (Ch) and the synthesis of acetylcholine (ACh) were measured in synaptosomes by measuring the utilization of [²H₄]Ch. The synthesis of ACh was reduced under several conditions which reduce the availability of acetyl coenzyme A (AcCoA) including no glucose added, replacement of glucose with succinate or impairment of glucose utilization by bromopyr-uvate, NaCN, or pentobarbital. These conditions did not reduce the amount of unacetylated [²H₄]Ch in the synaptosomes indicating that the high affinity transport of Ch is not directly coupled to the synthesis of ACh.     

On approaches to the functional restoration of salivary glands damaged by radiation therapy for head and neck cancer,with a review of related aspects of salivary gland morphology and development

Radiation therapy for cancer of the head and neck can devastate the salivary glands and partially devitalize the mandible and maxilla. As a result, saliva production is drastically reduced and its quality adversely altered. Without diligent home and professional care, the teeth are subject to rapid destruction by caries, necessitating extractions with attendant high risk of necrosis of the supporting bone. Innovative techniques in delivery of radiation therapy and administration of drugs that selectively protect normal tissues can reduce significantly the radiation effects on salivary glands. Nonetheless, many patients still suffer severe oral dryness. I review here the functional morphology and development of salivary glands as these relate to approaches to preventing and restoring radiation-induced loss of salivary function. The acinar cells are responsible for most of the fluid and organic material in saliva, while the larger ducts influence the inorganic content. A central theme of this review is the extent to which the several types of epithelial cells in salivary glands may be pluripotential and the circumstances that may influence their ability to replace cells that have been lost or functionally inactivated due to the effects of radiation. The evidence suggests that the highly differentiated cells of the acini and large ducts of mature glands can replace themselves except when the respective pools of available cells are greatly diminished via apoptosis or necrosis owing to severely stressful events. Under the latter circumstances, relatively undifferentiated cells in the intercalated ducts proliferate and redifferentiate as may be required to replenish the depleted pools. It is likely that some, if not many, acinar cells may de-differentiate into intercalated duct-like cells and thus add to the pool of progenitor cells in such situations. If the stress is heavy doses of radiation, however, the result is not only the death of acinar cells, but also a marked decline in functional differentiation and proliferative capacity of all of the surviving cells, including those with progenitor capability. Restoration of gland function, therefore, seems to require increasing the secretory capacity of the surviving cells, or replacing the acinar cells and their progenitors either in the existing gland remnants or with artificial glands.     

Second messengers regulate RGS2 expression which is targeted to the nucleus.

Regulators of G-protein Signaling (RGS) proteins attenuate signaling activities of G proteins, and modulation of expression appears to be a primary mechanism for regulating RGS proteins. In human astrocytoma 1321N1 cells RGS2 expression was increased by activation of muscarinic receptors coupled to phosphoinositide signaling with carbachol, or by increased cyclic AMP production, demonstrating that both signaling systems can increase the expression of a RGS family member in a single cell type. Carbachol-stimulated increases in endogenous RGS2 protein levels appeared by immunocytochemical analysis to be largely confined to the nucleus, and this localization was confirmed by Western blot analysis which showed increased nuclear, but not cytosolic, RGS2 after carbachol treatment. Additionally, transiently expressed green fluorescent protein (GFP)-tagged, 6xHis-tagged, or unmodified RGS2 resulted in a predominant nuclear localization, as well as a distinct accumulation of RGS2 along the plasma membrane. The intranuclear localization of GFP-RGS2 was confirmed with confocal microscopy. Thus, RGS2 expression is rapidly and transiently increased by phosphoinositide signaling and by cyclic AMP, and endogenous and transfected RGS2 is largely, although not entirely, localized in the nucleus.     

Calf thymus high mobility group proteins are nonenzymatically glycated but not significantly glycosylated

Over the past decade, there have been many reports suggesting the presence of complex carbohydrates on nuclear and cytoplasmic proteins in mammalian cells. Some of the most often cited of these reports deal with the glycosylation of the high mobility group (HMG) proteins. These are relatively abundant chromosomal proteins that are known to be associated with nucleosomes and actively transcribed regions of chromatin. The original report describing HMG protein glycosylation presented several lines of evidence suggesting that these proteins are glycosylated, including carbohydrate compositional analysis and periodic-acid Schiff staining. We have attempted to repeat these observations with more highly purified protein than was utilized in the original study. Using carbohydrate compositional analysis performed by high pH anion exchange chromatography coupled to pulsed-amperometric detection, we saw no evidence for significant glycosylation of these proteins. In addition, we found no evidence for the presence of O- GlcNAc, a well known form of nuclear glycosylation. The HMG proteins did react with periodate, suggesting the presence of a modification containing cis-diols on the protein. Several tryptic peptides isolated from HMG 14 and 17 which retained the periodate reactivity had in common lysine residues, suggesting a potential modification of the straightepsilon-amino groups of lysines such as nonenzymatic glycation. Western blot analysis of the HMG proteins using anti-advanced glycation endproducts (AGE) antibodies confirmed the presence of glycation products on the HMG proteins.