Characterization of the intronic splicing silencers flanking FGFR2 exon IIIb

The cell type-specific alternative splicing of FGFR2 pre-mRNA results in the mutually exclusive use of exons IIIb and IIIc, which leads to critically important differences in receptor function. The choice of exon IIIc in mesenchymal cells involves activation of this exon and repression of exon IIIb. This repression is mediated by the function of upstream and downstream intronic splicing silencers (UISS and DISS). Here we present a detailed characterization of the determinants of silencing function within UISS and DISS. We used a systematic mutational analysis, introducing deletions and substitutions to define discrete elements within these two silencers of exon IIIb. We show that UISS requires polypyrimidine tract-binding protein (PTB)-binding sites, which define the UISS1 sub-element, and an eight nucleotide sequence 5'-GCAGCACC-3' (UISS2) that is also required. Even though UISS2 does not bind PTB, the full UISS can be replaced with a synthetic silencer designed to provide optimal PTB binding. DISS is composed of a 5'-conserved sub-element (5'-CE) and two regions that contain multiple PTB sites and are functionally redundant (DISS1 and DISS2). DISS1 and DISS2 are separated by the activator sequence IAS2, and together these opposing elements form the intronic control element. Deletion of DISS in the FGFR2 exon IIIb context resulted in the near full inclusion of exon IIIb, and insertion of this silencer downstream of a heterologous exon with a weak 5' splice site was capable of repressing exon inclusion. Extensive deletion analysis demonstrated that the majority of silencing activity could be mapped to the conserved octamer CUCGGUGC within the 5'CE. Replacement of 5'CE and DISS1 with PTB-binding elements failed to restore repression of exon IIIb. We tested the importance of the relative position of the silencers and of the subelements within each silencer. Whereas UISS1, UISS2, DISS1, and DISS2 appear somewhat malleable, the 5'CE is rigid in terms of relative position and redundancy. Our data defined elements of function within the ISSs flanking exon IIIb and suggested that silencing of this exon is mediated by multiple trans-acting factors.     

Trimeric membrane-anchored gp41 inhibits HIV membrane fusion

The HIV-1 envelope glycoprotein is composed of a receptor binding subunit, gp120 that is non-covalently linked to the membrane-anchored fusion protein, gp41. Triggered by cellular receptor binding, the trimeric envelope complex mediates the fusion of viral and cellular membranes through the rearrangement of the fusion protein subunit into a six-helical bundle core structure. Here we describe the biophysical and functional properties of a membrane-anchored fragment of gp41 (gp41ctm) that includes the complete C-terminal heptad repeat region 2, the connecting part, and the transmembrane region. We show that the transmembrane domain of the envelope glycoprotein is sufficient for trimerization in vitro, contributing most of the alpha-helical content of gp41ctm. Trimeric gp41ctm is protease-resistant and recognizes neutralizing antibodies 2F5 and 4E10. However, gp41ctm and gp41ctm proteoliposomes elicit no clear neutralizing immune responses in preliminary mouse studies. We further show that gp41ctm and surprisingly also gp41ctm proteoliposomes have potent anti-viral activity. Our data suggest that liposome-anchored gp41ctm exerts its inhibitory action outside of the initial fusion contact site, and its implications for the fusion reaction are discussed.     

31P NMR and genetic analysis establish hinT as the only Escherchia coli purine nucleoside phosphoramidase and as essential for growth under high salt conditions

Eukaryotic cells encode AMP-lysine (AMP-N-epsilon-(N-alpha-acetyl lysine methyl ester) 5'-phosphoramidate) hydrolases related to the rabbit histidine triad nucleotide-binding protein 1 (Hint1) sequence. Bacterial and archaeal cells have Hint homologs annotated in a variety of ways, but the enzymes have not been characterized, nor have phenotypes been described due to loss of enzymatic activity. We developed a quantitative (31)P NMR assay to determine whether Escherichia coli possesses an adenosine phosphoramidase activity. Indeed, soluble lysates prepared from wild-type laboratory E. coli exhibited activity on the model substrate adenosine 5'-monophosphoramidate (AMP-NH(2)). The E. coli Hint homolog, which had been comprehensively designated ycfF and is here named hinT, was cloned, overexpressed, purified, and characterized with respect to purine nucleoside phosphoramidate substrates. Bacterial hinT was several times more active than human or rabbit Hint1 on five model substrates. In addition, bacterial and mammalian enzymes preferred guanosine versus adenosine phosphoramidates as substrates. Analysis of the lysates from a constructed hinT knock-out strain of E. coli demonstrated that all of the cellular purine nucleoside phosphoramidase activity is due to hinT. Physiological analysis of this mutant revealed that the loss of hinT results in failure to grow in media containing 0.75 m KCl, 0.9 m NaCl, 0.5 m NaOAc, or 10 mm MnCl(2). Thus, cation-resistant bacterial cell growth may be dependent on the hydrolysis of adenylylated and/or guanylylated phosphoramidate substrates by hinT.     

Nox4 NAD(P)H oxidase mediates hypertrophy and fibronectin expression in the diabetic kidney

Renal hypertrophy and extracellular matrix accumulation are early features of diabetic nephropathy. We investigated the role of the NAD(P)H oxidase Nox4 in generation of reactive oxygen species (ROS), hypertrophy, and fibronectin expression in a rat model of type 1 diabetes induced by streptozotocin. Phosphorothioated antisense (AS) or sense oligonucleotides for Nox4 were administered for 2 weeks with an osmotic minipump 72 h after streptozotocin treatment. Nox4 protein expression was increased in diabetic kidney cortex compared with non-diabetic controls and was down-regulated in AS-treated animals. AS oligonucleotides inhibited NADPH-dependent ROS generation in renal cortical and glomerular homogenates. ROS generation by intact isolated glomeruli from diabetic animals was increased compared with glomeruli isolated from AS-treated animals. AS treatment reduced whole kidney and glomerular hypertrophy. Moreover, the increased expression of fibronectin protein was markedly reduced in renal cortex including glomeruli of AS-treated diabetic rats. Akt/protein kinase B and ERK1/2, two protein kinases critical for cell growth and hypertrophy, were activated in diabetes, and AS treatment almost abolished their activation. In cultured mesangial cells, high glucose increased NADPH oxidase activity and fibronectin expression, effects that were prevented in cells transfected with AS oligonucleotides. These data establish a role for Nox4 as the major source of ROS in the kidneys during early stages of diabetes and establish that Nox4-derived ROS mediate renal hypertrophy and increased fibronectin expression.     

STIM1, an essential and conserved component of store-operated Ca2+ channel function

Store-operated Ca2+ (SOC) channels regulate many cellular processes, but the underlying molecular components are not well defined. Using an RNA interference (RNAi)-based screen to identify genes that alter thapsigargin (TG)-dependent Ca2+ entry, we discovered a required and conserved role of Stim in SOC influx. RNAi-mediated knockdown of Stim in Drosophila S2 cells significantly reduced TG-dependent Ca2+ entry. Patch-clamp recording revealed nearly complete suppression of the Drosophila Ca2+ release-activated Ca2+ (CRAC) current that has biophysical characteristics similar to CRAC current in human T cells. Similarly, knockdown of the human homologue STIM1 significantly reduced CRAC channel activity in Jurkat T cells. RNAi-mediated knockdown of STIM1 inhibited TG- or agonist-dependent Ca2+ entry in HEK293 or SH-SY5Y cells. Conversely, overexpression of STIM1 in HEK293 cells modestly enhanced TG-induced Ca2+ entry. We propose that STIM1, a ubiquitously expressed protein that is conserved from Drosophila to mammalian cells, plays an essential role in SOC influx and may be a common component of SOC and CRAC channels.     

Variogram analysis of the spatial genetic structure of continuous populations using multilocus microsatellite data

A geostatistical perspective on spatial genetic structure may explain methodological issues of quantifying spatial genetic structure and suggest new approaches to addressing them. We use a variogram approach to (i) derive a spatial partitioning of molecular variance, gene diversity, and genotypic diversity for microsatellite data under the infinite allele model (IAM) and the stepwise mutation model (SMM), (ii) develop a weighting of sampling units to reflect ploidy levels or multiple sampling of genets, and (iii) show how variograms summarize the spatial genetic structure within a population under isolation-by-distance. The methods are illustrated with data from a population of the epiphytic lichen Lobaria pulmonaria, using six microsatellite markers. Variogram-based analysis not only avoids bias due to the underestimation of population variance in the presence of spatial autocorrelation, but also provides estimates of population genetic diversity and the degree and extent of spatial genetic structure accounting for autocorrelation.     

Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells

PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.     

eIF4G and CBP80 share a common origin and similar domain organization: implications for the structure and function of eIF4G

Eukaryotic translation initiation factor 4G (eIF4G) plays a critical role in protein expression, and is at the center of a complex regulatory network. Together with the cap-binding protein eIF4E, it recruits the small ribosomal subunit to the 5'-end of mRNA and promotes the assembly of a functional translation initiation complex, which scans along the mRNA to the translation start codon. Human eIF4G contains three consecutive HEAT domains, as well as long unstructured regions involved in multiple protein-protein interactions. Despite the accumulating data about the structure and function of eIF4G, the mechanisms of coordination and regulation of its interactions with other factors have remained largely unknown. Here, we present evidence that eIF4G and the large subunit of the nuclear cap-binding complex, CBP80, share a common origin and domain structure. We propose that the organization of the individual domains in eIF4G and CBP80 could also be conserved. The structure of CBP80, in complex with the nuclear cap-binding protein CBP20, is used to build a model for the mutual orientation of the domains in eIF4G and their interactions with other factors. The organization of the CBP80-CBP20 complex suggests how the activity of eIF4G in translation initiation could be regulated through a dynamic network of overlapping intra- and intermolecular interactions centered around the eIF4G HEAT domains.     

Identification of the glycosylation site of the adenovirus type 5 fiber protein

The fiber protein purified from the pool of nonincorporated viral protein after infection of cells with adenovirus type 5 exists as two forms separable by reverse-phase HPLC. As determined by mass spectrometry, this heterogeneity results from a difference in one O-linked N-acetylglucosamine (GlcNac). A western blot analysis using a monoclonal antibody directed against the GlcNac motif showed that only one of the two forms reacted with the antibody, suggesting that one form carries a single GlcNac and the other form has none. The ratio of glycosylated to nonglycosylated forms of fiber, which is about 1, is conserved in assembled viruses. After digestion of glycosylated fiber with endoproteinase GluC, isolation of the glycosylated peptide by reverse-phase HPLC, and chemical derivatization using dimethylamine, the site of glycosylation was located in the fiber shaft at serine 109 by mass spectrometry. Elimination of glycosylation by site-directed mutagenesis of fiber should help to understand the function of this postranslational modification.     

Plasmacytoid dendritic cells control TLR7 sensitivity of naive B cells via type I IFN

Detailed information of human B cell activation via TLR may lead to a better understanding of B cell involvement in autoimmunity and malignancy. In this study we identified a fundamental difference in the regulation of TLR7- and TLR9-mediated B cell stimulation: whereas the induction of polyclonal naive B cell proliferation by the TLR7 ligands resiquimod (R848) and loxoribine required the presence of plasmacytoid dendritic cells (PDCs), activation via the TLR9 ligand CpG was independent of PDCs. We found that PDC-derived type I IFN enhanced TLR7 sensitivity of B cells by selectively up-regulating TLR7 expression. In contrast the expression levels of TLR9 and of other TLRs studied remained unchanged. In the presence of type I IFN, TLR7 ligation triggered polyclonal B cell expansion and B cell differentiation toward Ig-producing plasma cells; notably, this occurred independently of T cell help and B cell Ag. Human B cells did not respond to ligands of other TLRs including TLR2, TLR4 and TLR6 with and without type I IFN. In conclusion, our results reveal a distinct regulation of TLR7 and TLR9 function in human B cells and highlight TLR7 and TLR9 as unique targets for therapeutic intervention in B cell-mediated immunity and disease.