Double-stranded-RNA-dependent protein kinase and TAR RNA-binding protein form homo- and heterodimers in vivo.

The yeast two-hybrid system and far-Western protein blot analysis were used to demonstrate dimerization of human double-stranded RNA (dsRNA)-dependent protein kinase (PKR) in vivo and in vitro. A catalytically inactive mutant of PKR with a single amino acid substitution (K296R) was found to dimerize in vivo, and a mutant with a deletion of the catalytic domain of PKR retained the ability to dimerize. In contrast, deletion of the two dsRNA-binding motifs in the N-terminal regulatory domain of PKR abolished dimerization. In vitro dimerization of the dsRNA-binding domain required the presence of dsRNA. These results suggest that the binding of dsRNA by PKR is necessary for dimerization. The mammalian dsRNA-binding protein TRBP, originally identified on the basis of its ability to bind the transactivation region (TAR) of human immunodeficiency virus RNA, also dimerized with itself and with PKR in the yeast assay. Taken together, these results suggest that complexes consisting of different combinations of dsRNA-binding proteins may exist in vivo. Such complexes could mediate differential effects on gene expression and control of cell growth.

Dissociation between changes in cytoplasmic free Ca2+ concentration and insulin secretion as evidenced from measurements in mouse single pancreatic islets.

Simultaneous measurements of cytosolic free Ca2+ concentration and insulin release, in mouse single pancreatic islets, revealed a direct correlation only initially after stimulation with glucose or K+. Later, there is an apparent dissociation between these two parameters, with translocation of alpha and epsilon isoenzymes of protein kinase C to membranes and simultaneous desensitization of insulin release in response to glucose. Recovery of insulin release, without any concomitant changes in cytosolic free Ca2+ concentration, after addition of phorbol 12-myristate 13-acetate, okadaic acid, and forskolin supports the notion that the desensitization process is accounted for by dephosphorylation of key regulatory sites of the insulin exocytotic machinery.

An alternatively spliced form of the transcription factor Sp1 containing only a single glutamine-rich transactivation domain.

Protein-protein interactions involving specific transactivation domains play a central role in gene transcription and its regulation. The promoter-specific transcription factor Sp1 contains two glutamine-rich transcriptional activation domains (A and B) that mediate direct interactions with the transcription factor TFIID complex associated with RNA polymerase II and synergistic effects involving multiple Sp1 molecules. In the present study, we report the complementary DNA sequence for an alternatively spliced form of mouse Sp1 (mSp1-S) that lacks one of the two glutamine-rich activation regions present in the full-length protein. Corresponding transcripts were identified in mouse tissues and cell lines, and an Sp1-related protein identical in size to that predicted for mSp1-S was detected in mouse nuclear extracts. Cotransfection analysis revealed that mSp1-S lacks appreciable activity at promoters containing a single Sp1 response element but is active when multiple Sp1 sites are present, suggesting synergistic interactions between multiple mSp1-S molecules. The absence of a single glutamine-rich domain does not fully explain the properties of the smaller protein and indicates that additional structural features account for its unique transcriptional activity. The functional implications of this alternatively spliced form of Sp1 are discussed.

Single-headed myosin II acts as a dominant negative mutation in Dictyostelium.

Conventional myosin II is an essential protein for cytokinesis, capping of cell surface receptors, and development of Dictyostelium cells. Myosin II also plays an important role in the polarization and movement of cells. All conventional myosins are double-headed molecules but the significance of this structure is not understood since single-headed myosin II can produce movement and force in vitro. We found that expression of the tail portion of myosin II in Dictyostelium led to the formation of single-headed myosin II in vivo. The resultant cells contain an approximately equal ratio of double- and single-headed myosin II molecules. Surprisingly, these cells were completely blocked in cytokinesis and capping of concanavalin A receptors although development into fruiting bodies was not impaired. We found that this phenotype is not due to defects in myosin light chain phosphorylation. These results show that single-headed myosin II cannot function properly in vivo and that it acts as a dominant negative mutation for myosin II function. These results suggest the possibility that cooperativity of myosin II heads is critical for force production in vivo.

Oxidation of cysteine-322 in the repeat domain of microtubule-associated protein tau controls the in vitro assembly of paired helical filaments.

One of the hallmarks of Alzheimer disease is the pathological aggregation of tau protein into paired helical filaments (PHFs) and neurofibrillary tangles. Here we describe the in vitro assembly of recombinant tau protein and constructs derived from it into PHFs. Though whole tau assembled poorly, constructs containing three internal repeats (corresponding to the fetal tau isoform) formed PHFs reproducibly. This ability depended on intermolecular disulfide bridges formed by the single Cys-322. Blocking the SH group, mutating Cys for Ala, or keeping tau in a reducing environment all inhibited assembly. With constructs derived from four-repeat tau (having the additional repeat no. 2 and a second Cys-291), PHF assembly was blocked because Cys-291 and Cys-322 interact within the molecule. PHF assembly was enabled again by mutating Cys-291 for Ala. The synthetic PHFs bound the dye thioflavin S used in Alzheimer disease diagnostics. The data imply that the redox potential in the neuron is crucial for PHF assembly, independently or in addition to pathological phosphorylation reactions.

A yeast gene required for DNA replication encodes a protein with homology to DNA helicases.

A yeast gene has been identified by screening for DNA replication mutants using a permeabilized cell replication assay. The mutant is temperature sensitive for growth and shows a cell cycle phenotype typical of DNA replication mutants. RNA synthesis is normal in the mutant but DNA synthesis ceases upon shift to the nonpermissive temperature. The DNA2 gene was cloned by complementation of the dna2ts gene phenotype. The gene is essential for viability. The gene encodes a 172-kDa protein with characteristic DNA helicase motifs. A hemagglutinin epitope-Dna2 fusion protein was prepared and purified by conventional and immunoaffinity chromatography. The purified protein is a DNA-dependent ATPase and has 3' to 5' DNA helicase activity specific for forked substrates. A nuclease activity that endonucleolytically cleaves DNA molecules having a single-stranded 5' tail adjacent to a duplex region copurifies through all steps with the fusion protein.

A single GAL4 dimer can maximally activate transcription under physiological conditions.

Most eukaryotic promoters contain multiple binding sites for one or more transcriptional activators that interact in a synergistic manner. A common view is that synergism is a manifestation of the need for many contacts between activators and the general transcription machinery that a single activator presumably cannot fulfill. In this model, various combinations of protein-protein interactions control the level of gene expression. However, we show here that under physiological conditions, a single binding site and presumably GAL4 can activate transcription to the maximum possible level in vivo. Synergistic effects in this natural system are shown to be consistent with cooperative DNA binding. These results point to DNA occupancy as the major element in fine tuning gene expression in the galactose regulon.

Targeted disruption of the surfactant protein B gene disrupts surfactant homeostasis, causing respiratory failure in newborn mice.

Surfactant protein B (SP-B) is an 8.7-kDa, hydrophobic protein that enhances the spreading and stability of surfactant phospholipids in the alveolus. To further assess the role of SP-B in lung function, the SP-B gene was disrupted by homologous recombination in murine mouse embryonic stem cells. Mice with a single mutated SP-B allele (+/-) were unaffected, whereas homozygous SP-B -/- offspring died of respiratory failure immediately after birth. Lungs of SP-B -/- mice developed normally but remained atelectatic in spite of postnatal respiratory efforts. SP-B protein and mRNA were undetectable and tubular myelin figures were lacking in SP-B -/- mice. Type II cells of SP-B -/- mice contained no fully formed lamellar bodies. While the abundance of SP-A and SP-C mRNAs was not altered, an aberrant form of pro-SP-C, 8.5 kDa, was detected, and fully processed SP-C peptide was markedly decreased in lung homogenates of SP-B -/- mice. Ablation of the SP-B gene disrupts the routing, storage, and function of surfactant phospholipids and proteins, causing respiratory failure at birth.

Identification of receptor ligands and receptor subtypes using antagonists in a capillary electrophoresis single-cell biosensor separation system.

A capillary electrophoresis system with single-cell biosensors as a detector has been used to separate and identify ligands in complex biological samples. The power of this procedure was significantly increased by introducing antagonists that inhibited the cellular response from selected ligand-receptor interactions. The single-cell biosensor was based on the ligand-receptor binding and G-protein-mediated signal transduction pathways in PC12 and NG108-15 cell lines. Receptor activation was measured as increases in cytosolic free calcium ion concentration by using fluorescence microscopy with the intracellular calcium ion indicator fluo-3-acetoxymethyl ester. Specifically, a mixture of bradykinin (BK) and acetylcholine (ACh) was fractionated and the components were identified by inhibiting the cellular response with icatibant (HOE 140), a selective antagonist to the BK B2 receptor subtype (B2BK), and atropine, an antagonist to muscarinic ACh receptor subtypes. Structurally related forms of BK were also identified based on inhibiting B2BK receptors. Applications of this technique include identification of endogenous BK in a lysate of human hepatocellular carcinoma cells (Hep G2) and screening for bioactivity of BK degradation products in human blood plasma. The data demonstrate that the use of antagonists with a single-cell biosensor separation system aids identification of separated components and receptor subtypes.

Interaction of herpes simplex virus 1 origin-binding protein with DNA polymerase alpha.

The herpes simplex virus 1 (HSV-1) genome encodes seven polypeptides that are required for its replication. These include a heterodimeric DNA polymerase, a single-strand-DNA-binding protein, a heterotrimeric helicase/primase, and a protein (UL9 protein) that binds specifically to an HSV-1 origin of replication (oris). We demonstrate here that UL9 protein interacts specifically with the 180-kDa catalytic subunit of the cellular DNA polymerase alpha-primase. This interaction can be detected by immunoprecipitation with antibodies directed against either of these proteins, by gel mobility shift of an oris-UL9 protein complex, and by stimulation of DNA polymerase activity by the UL9 protein. These findings suggest that enzymes required for cellular DNA replication also participate in HSV-1 DNA replication.

Depletion-activated calcium current is inhibited by protein kinase in RBL-2H3 cells.

Whole-cell patch-clamp recordings and single-cell Ca2+ measurements were used to study the control of Ca2+ entry through the Ca2+ release-activated Ca2+ influx pathway (ICRAC) in rat basophilic leukemia cells. When intracellular inositol 1,4,5-trisphosphate (InsP3)-sensitive stores were depleted by dialyzing cells with high concentrations of InsP3, ICRAC inactivated only slightly in the absence of ATP. Inclusion of ATP accelerated inactivation 2-fold. The inactivation was increased further by the ATP analogue adenosine 5'-[gamma-thio]triphosphate, which is readily used by protein kinases, but not by 5'-adenylyl imidodiphosphate, another ATP analogue that is not used by kinases. Neither cyclic nucleotides nor inhibition of calmodulin or tyrosine kinase prevented the inactivation. Staurosporine and bisindolylmaleimide, protein kinase C inhibitors, reduced inactivation of ICRAC, whereas phorbol ester accelerated inactivation of the current. These results demonstrate that a protein kinase-mediated phosphorylation, probably through protein kinase C, inactivates ICRAC. Activation of the adenosine receptor (A3 type) in RBL cells did not evoke much Ca2+ influx or systematic activation of ICRAC. After protein kinase C was blocked, however, large ICRAC was observed in all cells and this was accompanied by large Ca2+ influx. The ability of a receptor to evoke Ca2+ entry is determined, at least in part, by protein kinase C. Antigen stimulation, which triggers secretion through a process that requires Ca2+ influx, activated ICRAC. The regulation of ICRAC by protein kinase will therefore have important consequences on cell functioning.

Generation of targeted retroviral vectors by using single-chain variable fragment: an approach to in vivo gene delivery.

We report the generation of a retroviral vector that infects human cells specifically through recognition of the low density lipoprotein receptor. The rationale for this targeted infection is to add onto the ecotropic envelope protein of Moloney murine leukemia virus, normally trophic for murine cells, a single-chain variable fragment derived from a monoclonal antibody recognizing the human low density lipoprotein receptor. This chimeric envelope protein was used to construct a packaging cell line producing a retroviral vector capable of high-efficiency transfer of the Escherichia coli beta-galactosidase gene to human cells expressing low density lipoprotein receptor. This approach offers a generalized plan to generate cell and tissue-specific retroviral vectors, an essential step toward in vivo gene therapy strategies.

Significance of bound water to local chain conformations in protein crystals.

We examine how the polypeptide chain in protein crystal structures exploits the multivalent hydrogen-bonding potential of bound water molecules. This shows that multiple interactions with a single water molecule tend to occur locally along the chain. A distinctive internal-coordinate representation of the local water-binding segments reveals several consensus conformations. The fractional water occupancy of each was found by comparison of the total number of conformations in the database regardless of the presence or absence of bound water. The water molecule appears particularly frequently in type II beta-turn geometries and an N-terminal helix feature. This work constitutes a first step into assessing not only the generality but also the significance of specific water binding in globular proteins.

Mucosal and systemic immune responses to a recombinant protein expressed on the surface of the oral commensal bacterium Streptococcus gordonii after oral colonization.

To circumvent the need to engineer pathogenic microorganisms as live vaccine-delivery vehicles, a system was developed which allowed for the stable expression of a wide range of protein antigens on the surface of Gram-positive commensal bacteria. The human oral commensal Streptococcus gordonii was engineered to surface express a 204-amino acid allergen from hornet venom (Ag5.2) as a fusion with the anchor region of the M6 protein of Streptococcus pyogenes. The immunogenicity of the M6-Ag5.2 fusion protein was assessed in mice inoculated orally and intranasally with a single dose of recombinant bacteria, resulting in the colonization of the oral/pharyngeal mucosa for 10-11 weeks. A significant increase of Ag5.2-specific IgA with relation to the total IgA was detected in saliva and lung lavages when compared with mice colonized with wild-type S. gordonii. A systemic IgG response to Ag5.2 was also induced after oral colonization. Thus, recombinant Gram-positive commensal bacteria may be a safe and effective way of inducing a local and systemic immune response.

A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity.

Construction of a bispecific single-chain antibody derivative is described that consists of two different single-chain Fv fragments joined through a Gly-Ser linker. One specificity of the two Fv fragments is directed against the CD3 antigen of human T cells and the other is directed against the epithelial 17-1A antigen; the latter had been found in a clinical trial to be a suitable target for antibody therapy of minimal residual colorectal cancer. The construct could be expressed in CHO cells as a fully functional protein, while its periplasmic expression in Escherichia coli resulted in a nonfunctional protein only. The antigen-binding properties of the bispecific single-chain antibody are indistinguishable from those of the corresponding univalent single-chain Fv fragments. By redirecting human peripheral T lymphocytes against 17-1A-positive tumor cells, the bispecific antibody proved to be highly cytotoxic at nanomolar concentrations as demonstrated by 51Cr release assay on various cell lines. The described bispecific construct has a molecular mass of 60 kDa and can be easily purified by its C-terminal histidine tail on a Ni-NTA chromatography column. As bispecific antibodies have already been shown to be effective in vivo in experimental tumor systems as well as in phase-one clinical trials, the small CD3/17-1A-bispecific antibody may be more efficacious than intact antibodies against minimal residual cancer cells.