O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. Beta-elimination/Michael addition, MS(3) on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including "PVST" and a novel "TTA" motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.

Heat shock induced gene expression and other cellular responses help limit the damage caused by stress and thus facilitate cellular recovery. Cellular damage also triggers apoptotic cell death through several pathways. This paper briefly reviews interactions of the major heat shock proteins with components of the apoptotic pathways. Hsp90, which acts as a chaperone for unstable signal transducers to keep them poised for activation, interacts with RIP and Akt and promotes NF-kappa B mediated inhibition of apoptosis; in addition it also blocks some steps in the apoptotic pathways. Hsp70 is mostly anti-apoptotic and acts at several levels like inhibition of translocation of Bax into mitochondria, release of cytochrome c from mitochondria,formation of apoptosome and inhibition of activation of initiator caspases. Hsp70 also modulates JNK,NF-kappa B and Akt signaling pathways in the apoptotic cascade. In contrast, Hsp60 has both anti-and pro-apoptotic roles. Cytosolic Hsp60 prevents translocation of the pro-apoptotic protein Bax into mitochondria and thus promotes cell survival but it also promotes maturation of procaspase-3,essential for caspase mediated cell death. Our recent in vivo studies show that RNAi for the Hsp60D in Drosophila melanogaster prevents induced apoptosis. Hsp27 exerts its anti-apoptotic influence by inhibiting cytochrome c and TNF-mediated cell death. alpha beta crystallin suppresses caspase-8 and cytochrome c mediated activation of caspase-3. Studies in our laboratory also reveal that absence or reduced levels of the developmentally active as well as stress induced non-coding hsr omega transcripts, which are known to sequester diverse hnRNPs and related nuclear RNA-binding proteins,block induced apoptosis in Drosophila. Modulation of the apoptotic pathways by Hsps reflects their roles as "weak links" between various "hubs" in cellular networks. On the other hand, non-coding RNAs, by virtue of their potential to bind with multiple proteins,can act as "hubs" in these networks. In view of the integrative nature of living systems, it is not surprising that stress-induced genes,generally believed to primarily function in cell survival pathways, inhibit or even promote cell death pathways at multiple levels to ensure homeostasis at cell and/or organism level. The heat shock genes obviously do much more than merely help cells survive stress.

Drugs that act on dopamine neurotransmission are important tools for the management of multiple neuropsychiatric disorders. Classically, dopamine receptors have been shown to regulate cAMP-PKA (protein kinase A) and Ca(2+) pathways through G-protein-mediated signaling. However, it has become apparent that, in addition to this canonical action, D(2)-class dopamine receptors can function through a protein kinase B (Akt)-GSK-3 (glycogen synthase kinase 3) signaling cascade. This novel signaling mode involves the multifunctional scaffolding protein beta-arrestin 2, which has a role in G-protein-coupled receptor (GPCR) desensitization. In this article, we provide an overview of how this dual function of components of the GPCR desensitization machinery relates to dopamine-receptor-mediated responses and we summarize recent insights into the relevance of the Akt-GSK-3 signaling cascade for the expression of dopamine-associated behaviors and the actions of dopaminergic drugs.

Among 21 different polysaccharides tested, 5 greatly enhanced the spontaneous and cyclic AMP-induced formation of exolipase: glycogen, hyaluronate, laminarin, pectin B, and gum arabic. These polysaccharides have in common the tendency to form highly ordered networks because of the branching or helical arrangement, or both, of their molecules. None of the polysaccharides could be utilized by the cells as the sole carbon source. Strong lipid extraction of four different polysaccharides did not reduce their exolipase-enhancing efficacy. At a constant cell density the stimulation of exolipase formation by various concentrations of glycogen followed saturation kinetics, suggesting a limited number of "sites" for the glycogen to act. The active principle present in a solution of pectin was destroyed by degradation (beta-elimination) of the polymer. Hyaluronate lost its exolipase-enhancing activity by exhaustive hydrolysis with hyaluronidase but was resistant to proteinase K. Exopolysaccharide, isolated from growth medium of Serratia marcescens SM-6, enhanced the exolipase formation as efficiently as hyaluronate. The results of this work are discussed mainly in terms of the "detachment hypothesis."

Using freshly isolated Ia+ gut epithelial cells we have been able to demonstrate that these cells can function as accessory cells in an immune response. The cells can act as stimulators in both autologous and allogeneic MLRs. More importantly, these cells are capable of taking up the soluble antigen, tetanus toxoid, processing it, and presenting it to tetanus-primed T cells. These functions appear to relate to the presence of surface Ia in that a hetero-anti-Ia antibody can block these effects. Noteworthy is the finding that the subpopulation of T cells stimulated when epithelial cells are used as accessory cells is the T8+, 9.3-T cell. These cells function as potent antigen-nonspecific suppressor cells in both MLR, T cell antigen responses, and induction of B cell differentiation by PWM. These findings have significant implications in local gut immune responses and may help explain several poorly characterized phenomena of mucosal immunity.

A study of papers on amyloid fibers suggested to us that cylindrical beta-sheets are the only structures consistent with some of the x-ray and electron microscope data. We then found that our own 7-year-old and hitherto enigmatic x-ray diagram of poly-L-glutamine fits a cylindrical sheet of 31 A diameter made of beta-strands with 20 residues per helical turn. Successive turns are linked by hydrogen bonds between both the main chain and side chain amides, and side chains point alternately into and out of the cylinder. Fibers of the exon-1 peptide of huntingtin and of the glutamine- and asparagine-rich region of the yeast prion Sup35 give the same underlying x-ray diagrams, which show that they have the same structure. Electron micrographs show that the 100-A-thick fibers of the Sup35 peptide are ropes made of three protofibrils a little over 30 A thick. They have a measured mass of 1,450 Da/A, compared with 1,426 Da/A for a calculated mass of three protofibrils each with 20 residues per helical turn wound around each other with a helical pitch of 510 A. Published x-ray diagrams and electron micrographs show that fibers of synuclein, the protein that forms the aggregates of Parkinson disease, consist of single cylindrical beta-sheets. Fibers of Alzheimer A beta fragments and variants are probably made of either two or three concentric cylindrical beta-sheets. Our structure of poly-L-glutamine fibers may explain why, in all but one of the neurodegenerative diseases resulting from extension of glutamine repeats, disease occurs when the number of repeats exceeds 37-40. A single helical turn with 20 residues would be unstable, because there is nothing to hold it in place, but two turns with 40 residues are stabilized by the hydrogen bonds between their amides and can act as nuclei for further helical growth. The A beta peptide of Alzheimer's disease contains 42 residues, the best number for nucleating further growth. All these structures are very stable; the best hope for therapies lies in preventing their growth.

The regulation of tyrosine phosphorylation and associated signalling through antigen, growth-factor and cytokine receptors is mediated by the reciprocal activities of protein tyrosine kinases and protein tyrosine phosphatases (PTPases). The transmembrane PTPase CD45 is a key regulator of antigen receptor signalling in T and B cells. Src-family kinases have been identified as primary molecular targets for CD45 (ref. 4). However, CD45 is highly expressed in all haematopoietic lineages at all stages of development, indicating that CD45 could regulate other cell types and might act on additional substrates. Here we show that CD45 suppresses JAK (Janus kinase) kinases and negatively regulates cytokine receptor signalling. Targeted disruption of the cd45 gene leads to enhanced cytokine and interferon-receptor-mediated activation of JAKs and STAT (signal transducer and activators of transcription) proteins. In vitro, CD45 directly dephosphorylates and binds to JAKs. Functionally, CD45 negatively regulates interleukin-3-mediated cellular proliferation, erythropoietin-dependent haematopoieisis and antiviral responses in vitro and in vivo. Our data identify an unexpected and novel function for CD45 as a haematopoietic JAK phosphatase that negatively regulates cytokine receptor signalling.

Smad proteins have been identified as mediators of intracellular signal transduction by members of the transforming growth factor-beta (TGF-beta) superfamily, which affect cell proliferation, differentiation, as well as pattern formation during early vertebrate development. Following receptor activation, Smads are assembled into heteromeric complexes consisting of a pathway-restricted Smad and the common Smad4 that are subsequently translocated into the nucleus where they are thought to play an important role in gene transcription. Here we report the identification of Smad Binding Elements (SBEs) composed of the sequence CAGACA in the promoter of the JunB gene, an immediate early gene that is potently induced by TGF-beta, activin, and bone morphogenetic protein (BMP) 2. Two JunB SBEs are arranged as an inverted repeat that is transactivated in response to Smad3 and Smad4 co-overexpression and shows inducible binding of a Smad3- and Smad4-containing complex in nuclear extracts from TGF-beta-treated cells. Bacterial-expressed Smad proteins bind directly to the SBE. Multimerization of the SBE creates a powerful TGF-beta-inducible enhancer that is also responsive to activin and BMPs. The identification of the sequence CAGACA as a direct binding site for Smad proteins will facilitate the identification of regulatory elements in genes that are activated by members of the TGF-beta superfamily.

One of the earliest cell surface antigens expressed by T cells following activation is CD69, which is detectable within one h of ligation of the T cell receptor/CD3 complex. Once expressed, CD69 acts as a costimulatory molecule for T cell activation and proliferation. In addition to mature T cells, CD69 is inducibly expressed by immature thymocytes, B cells, natural killer (NK) cells, monocytes, neutrophils and eosinophils, and is constitutively expressed by mature thymocytes and platelets. Recently, cDNA clones encoding human and mouse CD69 were isolated and showed CD69 to be a member of the C-type lectin superfamily. Gene mapping studies have placed CD69 on distal mouse chromosome 6 and human chromosome 12p13, close to, if not in, the NK gene complex. The structure, chromosomal localization, expression and function of CD69 suggest that it is likely a pleiotropic immune regulator, potentially important not only in NK cell function but also in the activation and differentiation of a wide variety of hematopoietic cells.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is an inducible cell adhesion glycoprotein of the immunoglobulin supergene family expressed on the surface of a wide variety of cell types. ICAM-1 interactions with the beta2 integrins CD11a/CD18 (LFA-1) and CD11b/CD18 (MAC-1) on the surface of leukocytes are important for their transendothelial migration to sites of inflammation and their function as costimulatory molecules for T cell activation. ICAM-1 is constitutively expressed on the cell surface and is up-regulated in response to a variety of inflammatory mediators, including proinflammatory cytokines, hormones, cellular stresses, and virus infection. These stimuli increase ICAM-1 expression primarily through activation of ICAM-1 gene transcription. During the past decade much has been learned about the cell type- and stimulus-specific transcription of ICAM-1. The architecture of the ICAM-1 promoter is complex, containing a large number of binding sites for inducible transcription factors, the most important of which is nuclear factor-kappa B (NF-kappaB). NF-kappaB acts in concert with other transcription factors and co-activators via specific protein-protein interactions, which facilitate the assembly of distinct stereospecific transcription complexes on the ICAM-1 promoter. These transcription complexes presumably mediate the induction of ICAM-1 expression in different cell types and in response to different stimuli. In this review, we summarize our current understanding of ICAM-1 gene regulation with a particular emphasis on the transcription factors and signal transduction pathways critical for the cell type- and stimulus-specific activation of ICAM-1 gene transcription.

BCAAs stimulate protein synthesis in in vitro preparations of skeletal muscle. Likewise, the stimulation of protein synthesis in skeletal muscle produced by intake of a mixed meal is due largely to BCAAs. Of the three BCAAs, leucine is the one primarily responsible for the stimulation of protein synthesis under these circumstances. The stimulatory effect of leucine on protein synthesis is mediated through upregulation of the initiation of mRNA translation. A number of mechanisms, including phosphorylation of ribosomal protein S6 Kinase, eukaryotic initiation factor (eIF)4E binding protein-1, and eIF4G, contribute to the effect of leucine on translation initiation. These mechanisms not only promote global translation of mRNA but also contribute to processes that mediate discrimination in the selection of mRNA for translation. A key component in a signaling pathway controlling these phosphorylation-induced mechanisms is the protein kinase, termed the mammalian target of rapamycin (mTOR). The activity of mTOR toward downstream targets is controlled in part through its interaction with the regulatory-associated protein of mTOR (known as raptor) and the G protein beta-subunit-like protein. Signaling through mTOR is also controlled by upstream members of the pathway such as the Ras homolog enriched in brain (Rheb), a GTPase that activates mTOR, and tuberin (also known as TSC2), a GTPase-activating protein, which, with its binding partner hamartin (also known as TSC1), acts to repress mTOR. Candidates for mediating the action of leucine to stimulate signaling through the mTOR pathway include TSC2, Rheb, and raptor. The current state of our understanding of how leucine acts on these signaling pathways and molecular mechanisms to stimulate protein synthesis in skeletal muscle is summarized in this article.

Traditionally, the ketone body β-hydroxybutyrate (βOHB) has been looked upon as a carrier of energy from liver to peripheral tissues during fasting or exercise. However, βOHB also signals via extracellular receptors and acts as an endogenous inhibitor of histone deacetylases (HDACs). These recent findings support a model in which βOHB functions to link the environment, in this case the diet, and gene expression via chromatin modifications. We review the regulation and functions of ketone bodies, the relationship between ketone bodies and calorie restriction, and the implications of HDAC inhibition by the ketone body βOHB in the modulation of metabolism and in diseases of aging.

Hepadnaviruses, including human hepatitis B virus (HBV), replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Despite this kinship to retroviruses, there are fundamental differences beyond the fact that hepadnavirions contain DNA instead of RNA. Most peculiar is the initiation of reverse transcription: it occurs by protein-priming, is strictly committed to using an RNA hairpin on the pgRNA, epsilon, as template, and depends on cellular chaperones; moreover, proper replication can apparently occur only in the specialized environment of intact nucleocapsids. This complexity has hampered an in-depth mechanistic understanding. The recent successful reconstitution in the test tube of active replication initiation complexes from purified components, for duck HBV (DHBV), now allows for the analysis of the biochemistry of hepadnaviral replication at the molecular level. Here we review the current state of knowledge at all steps of the hepadnaviral genome replication cycle, with emphasis on new insights that turned up by the use of such cell-free systems. At this time, they can, unfortunately, not be complemented by three-dimensional structural information on the involved components. However, at least for the epsilon RNA element such information is emerging, raising expectations that combining biophysics with biochemistry and genetics will soon provide a powerful integrated approach for solving the many outstanding questions. The ultimate, though most challenging goal, will be to visualize the hepadnaviral reverse transcriptase in the act of synthesizing DNA, which will also have strong implications for drug development.

Transforming growth factor beta (TGF beta) and activin each bind to pairs of membrane proteins, known as receptor types I and II, that associate to form a signaling complex. We report that TSR-I and ActR-I, two human transmembrane serine/threonine kinases distantly related to TGF beta and activin type II receptors, act as type I receptors for these factors. TSR-I is a type I receptor shared by TGF beta and activin, whereas ActR-I is an activin type I receptor. ActR-I, but not TSR-I, signals a particular transcriptional response in concert with activin type II receptors. The results indicate that type I receptors are transmembrane protein kinases that associate with type II receptors to generate diverse heteromeric serine/threonine kinase complexes of different signaling capacities.

The suppressive capacity of naturally occurring mouse CD4+CD25+ T cells on T-cell activation has been well documented. The present study is focused on the interaction of CD4+CD25+ T cells and B cells. By coculturing preactivated CD4+CD25+ T cells with B cells in the presence of polyclonal B-cell activators, we found that B-cell proliferation was significantly suppressed. The suppression of B-cell proliferation was due to increased cell death caused by the CD4+CD25+ T cells in a cell-contact-dependent manner. The induction of B-cell death is not mediated by Fas-Fas ligand pathway, but surprisingly, depends on the up-regulation of perforin and granzymes in the CD4+CD25+ T cells. Furthermore, activated CD4+CD25+ T cells preferentially killed antigen-presenting but not bystander B cells. Our results demonstrate that CD4+CD25+ T cells can act directly on B cells and suggest that the prevention of autoimmunity by CD4+CD25+ T cells can be explained, at least in part, by the direct regulation of B-cell function.

Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/β hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.

Staphylococcal enterotoxins (SE) are the most potent mitogens for T lymphocytes known; concentrations of less than 10(-9) M are sufficient for T cell activation. The mechanism of T cell activation by SE is unknown. We have used cloned human cytotoxic and proliferative T lymphocytes to dissect the molecular mechanism of T cell activation by SE. With rare exceptions, all TCR alpha/beta chain-expressing T cell clones of CD4+ or CD8+ phenotype, as well as CD4-8- TCR alpha/beta chain negative chain-expressing T lymphocyte clones, respond with proliferation and/or cytotoxicity to SE. For triggering of all these clones, the presence of autologous or allogeneic MHC class II molecules on accessory or target cells is necessary. This requirement for class II antigens is not due to an immunological recognition of processed SE, since inhibition of antigen processing has no influence on the T cell response to SE. SE acts on the T cells directly since (a) they stimulate a rise in intracellular calcium concentration in T cell lines or purified T cells, and (b) accessory cells can be replaced by phorbolesters in the proliferative activation of resting T cells by SE. Furthermore, the T cell response to SE shows extensive clonal heterogeneity. These results suggest that SE are functionally bivalent mitogens binding highly selectively to HLA class II molecules and the TCR. Thus, compared with other polyclonal T cell activating agents, activation with SE most closely mimicks the physiological way of MHC-restricted antigen recognition by T lymphocytes.

Metazoan spliceosomes exhibit an elaborate protein composition required for canonical and alternative splicing. Thus, the minimal set of proteins essential for activation and catalysis remains elusive. We therefore purified in vitro assembled, precatalytic spliceosomal complex B, activated B(act), and step 1 complex C from the simple eukaryote Saccharomyces cerevisiae. Mass spectrometry revealed that yeast spliceosomes contain fewer proteins than metazoans and that each functional stage is very homogeneous. Dramatic compositional changes convert B to B(act), which is composed of approximately 40 evolutionarily conserved proteins that organize the catalytic core. Additional remodeling occurs concomitant with step 1, during which nine proteins are recruited to form complex C. The moderate number of proteins recruited to complex C will allow investigations of the chemical reactions in a fully defined system. Electron microscopy reveals high-quality images of yeast spliceosomes at defined functional stages, indicating that they are well-suited for three-dimensional structure analyses.

Recent demonstrations of survival-promoting activity by neurotrophic agents in diverse neuronal systems have raised the possibility of pharmacological therapy for inherited and degenerative disorders of the central nervous system. We have shown previously that, in the retina, basic fibroblast growth factor delays photoreceptor degeneration in Royal College of Surgeons rats with inherited retinal dystrophy and that the growth factor reduces or prevents the rapid photoreceptor degeneration produced by constant light in the rat. This light-damage model now provides an efficient way to assess quantitatively the survival-promoting activity in vivo of a number of growth factors and other molecules. We report here that photoreceptors can be significantly protected from the damaging effects of light by intravitreal injection of eight different growth factors, cytokines, and neurotrophins that typically act through several distinct receptor families. In addition to basic fibroblast growth factor, those factors providing a high degree of photoreceptor rescue include brain-derived neurotrophic factor, ciliary neurotrophic factor, interleukin 1 beta, and acidic fibroblast growth factor; those with less activity include neurotrophin 3, insulin-like growth factor II, and tumor necrosis factor alpha; those showing little or no protective effect are nerve growth factor, epidermal growth factor, platelet-derived growth factor, insulin, insulin-like growth factor I, heparin, and laminin. Although we used at least one relatively high concentration of each agent (the highest available), it is still possible that other concentrations or factor combinations might be more protective. Injecting heparin along with acidic fibroblast growth factor or basic fibroblast growth factor further enhanced the degree of photoreceptor survival and also suppressed the increased incidence of macrophages produced by either factor, especially basic fibroblast growth factor. These results now provide the impetus for determining the normal function in the retina, mechanism(s) of rescue, and therapeutic potential in human eye diseases for each agent.

Anteroposterior (AP) patterning of the vertebrate neural plate is initiated during gastrulation and is regulated by Spemann's organizer and its derivatives. The prevailing model for AP patterning predicts a caudally increasing gradient of a 'transformer' which posteriorizes anteriorly specified neural cells. However, the molecular identity of the transforming gradient has remained elusive. We show that in Xenopus embryos (1) dose-dependent Wnt signalling is both necessary and sufficient for AP patterning of the neuraxis, (2) Wnt/beta-catenin signalling occurs in a direct and long-range fashion within the ectoderm, and (3) that there is an endogenous AP gradient of Wnt/beta-catenin signalling in the presumptive neural plate of the Xenopus gastrula. Our results indicate that an activity gradient of Wnt/beta-catenin signalling acts as transforming morphogen to pattern the Xenopus central nervous system.

Members of the Wnt/Wingless (Wg) family of signalling proteins organize many aspects of animal development by regulating the expression of particular target genes in responding cells. Recent biochemical studies indicate that the vertebrate HMG-domain proteins Lef-1 and XTcf-3 can physically interact with beta-catenin, a homologue of Drosophila Armadillo (Arm), the most downstream component known in the Wnt signal transduction pathway. However, these studies do not address whether the endogenous Lef/Tcf family members are required in vivo to transduce Wnt signals. Using genetic methods in Drosophila, we define a new segment polarity gene, pangolin (pan), and show that its product is required in vivo for Wg signal transduction in embryos and in developing adult tissues. In addition, we show that pan encodes a Lef/Tcf homologue and provide evidence that its protein product binds to the beta-catenin homologue Armadillo in vivo. Finally, we demonstrate that Pan functions downstream of Arm to transduce the Wg signal. Thus, our results indicate that Pan is an essential component of the Wg transduction pathway and suggest that it acts directly to regulate gene transcription in response to Wg signalling.

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, beta-transducin repeat-containing protein (betaTrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a betaTrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes.

Type I (alpha, beta) and type II (gamma) interferons (IFNs) can restrict the growth of many cell types. INF-stimulated gene transcription, a key early event in IFN response, acts through the Janus kinase-signal transducers and activators of transcription pathway, in which both IFN-alpha and IFN-gamma activate the transcription factor Stat1. A cell line lacking Stat1 (U3A) was not growth-arrested by IFN-alpha or IFN-gamma, and experiments were carried out with U3A cells permanently expressing normal or various mutant forms of Stat1 protein. Only cells in which complete Stat1 activity was available (Stat1alpha) were growth-inhibited by IFN-gamma. A mutant that supports 20-30% normal transcription did not cause growth restraint. In contrast, IFN-alpha growth restraint was imposed by cells producing Stat1beta, which lacks transcriptional activation potential. This parallels earlier results showing the truncated Stat1 can function in IFN-alpha gene activation. In addition to experiments on long-term cultured cells, we also found that wild-type primary mouse embryonic fibroblasts were inhibited by IFNs, but fibroblasts from Stat1-deficient mouse embryos were not inhibited by IFNs.

Iterative profile searches and structural modeling show that bacterial DnaG-type primases, small primase-like proteins from bacteria and archaea, type IA and type II topoisomerases, bacterial and archaeal nucleases of the OLD family and bacterial DNA repair proteins of the RecR/M family contain a common domain, designated Toprim (topoisomerase-primase) domain. The domain consists of approximately 100 amino acids and has two conserved motifs, one of which centers at a conserved glutamate and the other one at two conserved aspartates (DxD). Examination of the structure of Topo IA and Topo II and modeling of the Toprim domains of the primases reveal a compact beta/alpha fold, with the conserved negatively charged residues juxtaposed, and inserts seen in Topo IA and Topo II. The conserved glutamate may act as a general base in nucleotide polymerization by primases and in strand rejoining by topoisomerases and as a general acid in strand cleavage by topoisomerases and nucleases. The role of this glutamate in catalysis is supported by site-directed mutagenesis data on primases and Topo IA. The DxD motif may coordinate Mg2+that is required for the activity of all Toprim-containing enzymes. The common ancestor of all life forms could encode a prototype Toprim enzyme that might have had both nucleotidyl transferase and polynucleotide cleaving activity.