Bovine adenovirus type 3 internalization is independent of primary receptors of human adenovirus type 5 and porcine adenovirus type 3

Usefulness of adenoviral vectors derived from human adenovirus (HAd) type 5 (HAd5) is mainly limited by wide prevalence of preexisting anti-HAd5 immunity as well as non-specific tissue tropism of these vectors. As an alternative, non-human adenoviral vectors including bovine adenovirus type 3 (BAd3) are currently being investigated. Non-prevalence of BAd3 in humans and its ability to evade preexisting HAd immunity are some of the features that make BAd3 a promising vector for human gene delivery. BAd3 appears to have a tissue tropism distinct from that of HAd5 and also the repertoire of cells efficiently transduced by BAd3 is different. We performed antibody-mediated receptor blocking experiments to show that BAd3 internalization was independent of coxsackievirus-adenovirus receptor, the primary determinant of HAd5 tropism, or integrin alpha(v)beta3, a secondary molecule involved in HAd5 entry. Using homologous and heterologous knob-mediated competition assays with recombinant knobs of HAd5, porcine adenovirus type 3 (PAd3), or BAd3, we observed that BAd3 internalization was independent of the primary receptors of HAd5 and PAd3. These results provide support for further exploration of BAd3 vectors for designing targeted vectors for human gene therapy.     

Bob1, a Gim5/MM-1/Pfd5 homolog, interacts with the MAP kinase kinase Byr1 to regulate sexual differentiation in the fission yeast, Schizosaccharomyces pombe

The MAPKK Byr1 is an essential component of a Ras-dependent MAPK module required for sexual differentiation in the fission yeast, Schizosaccharomyces pombe. Here we describe the genetic and molecular characterization of a highly conserved protein, Bob1, which was identified from a two-hybrid screen for Byr1-interacting proteins. Byrl and Bobl proteins coprecipitate from S. pombe cell lysates, and both proteins localize to the tips and septa of S. pombe cells. S. pombe bob1 null (bob1delta) mutants lack obvious growth defects but exhibit a significant mating deficiency, which can be suppressed by overexpression of Byrl. Overexpression of Bob1 also leads to inhibition of mating in S. pombe, and this defect is likewise suppressed by Byrl overexpression. Bob1 is highly homologous in structure to the mammalian MM-1/Pfd5 and budding yeast Gim5/Pfd5-Sc proteins, which have been implicated as regulators of actin and tubulins. Similar to budding yeast gim5/pfd5-Sc mutants, S. pombe bob1delta cells have cytoskeletal defects, as judged by hypersensitivity to cytoskeletal disrupting drugs. byr1delta mutants do not share this characteristic with bob1delta mutants, and byr1delta bob1delta mutants are not significantly more sensitive to cytoskeletal disrupting drugs than cells carrying only the bob1delta mutation. Taken together, our results suggest that Bob1 has Byr1-related function(s) required for proper mating response of S. pombe cells and Byrl-independent function(s) required for normal cytoskeletal control. We show that the human MM-1/Pfd5 protein can substitute for its counterpart in fission yeast, providing evidence that the functions of Bob1-related proteins have been highly conserved through evolution. Our results lead us to propose that Bob1-related proteins may play diverse roles in eukaryotic organisms.     

Hyaluronan suppresses epidermal differentiation in organotypic cultures of rat keratinocytes

Hyaluronan (hyaluronic acid, HA) is an abundant matrix component between keratinocytes of the epidermis in vivo, but its function there remains unclear. We used a lift culture model, in which rat epidermal keratinocytes (REKs) stratify at an air-liquid interface, to ask whether HA may regulate epidermal proliferation and/or differentiation. In this model, early markers of differentiation (keratin 10), and later markers (profilaggrin, keratohyalin granules, cornified layers) are faithfully expressed, both temporally and spatially. HA, measured using two different analytical techniques, accumulated to high levels only in the presence of an intact basement membrane that seals the epidermal compartment. To test whether HA has a functional role in differentiation, Streptomyces hyaluronidase (StrepH, 1 U/ml; digests >95% of HA within 4 h) was added daily to lift cultures during stratification time-course experiments over 5 days. In StrepH-treated cultures, the expression of profilaggrin and the number and size of keratohyalin granules were significantly increased relative to controls using semiquantitative histological analyses. The StrepH-related accumulation of K10 protein and profilaggrin/filaggrin were confirmed by Western analyses. Thus, it appears that the presence of intercellular HA in the epidermis acts as a brake upon intracellular events that occur during keratinocyte differentiation.     

Influence of malaria parasite (P. vivax) on erythrocyte aggregation: a study based on dynamic imaging and analysis

Malaria due to P. vivax (PV) is prevalent in many countries. The present work is aimed to determine the cell-cell interaction through formation of aggregates under dynamic conditions. Blood samples are obtained from patients (n=11) suffering from PV malaria, and the normal subjects (n=10) in test tubes containing citrate phosphate dextrose (10:1.4), as an anticoagulant. The signature analysis of infected erythrocytes shows significant alterations in their shape and membrane. For aggregation analysis, erythrocyte suspension in plasma at hematocrit 5%, was placed in a glass chamber and mounted vertically on the stage of the video-microscope system. The aggregate images thus acquired show erythrocytes adhering with each other to form mash-like structures. With increase in parasitaemia, the erythrocytes show hyper-aggregation compared to that of normal cells. By processing of the sequence of recorded images during sedimentation, the various aggregation parameters are obtained. These parameters show that the formed aggregates are compact which produce distinct changes in sedimentation pattern with significantly higher sedimentation velocity compared to that in healthy blood samples. These changes in malaria could partly be responsible for alteration in blood flow through microcirculatory system.     

Crystallographic and biochemical analysis of cocaine-degrading antibody 15A10

Catalytic antibody 15A10 hydrolyzes the benzoyl ester of cocaine to form the nonpsychoactive metabolites benzoic acid and ecgonine methylester. Here, we report biochemical and structural studies that characterize the catalytic mechanism. The crystal structure of the cocaine-hydrolyzing monoclonal antibody (mAb) 15A10 has been determined at 2.35 A resolution. The binding pocket is fairly shallow and mainly hydrophobic but with a cluster of three hydrogen-bond donating residues (TrpL96, AsnH33, and TyrH35). Computational docking of the transition state analogue (TSA) indicates that these residues are appropriately positioned to coordinate the phosphonate moiety of the TSA and, hence, form an oxyanion hole. Tyrosine modification of the antibody with tetranitromethane reduced hydrolytic activity to background level. The contribution from these and other residues to catalysis and TSA binding was explored by site-directed mutagenesis of 15A10 expressed in a single chain fragment variable (scFv) format. The TyrH35Phe mutant had 4-fold reduced activity, and TrpL96Ala, TrpL96His, and AsnH33Ala mutants were all inactive. Comparison with an esterolytic antibody D2.3 revealed a similar arrangement of tryptophan, asparagine, and tyrosine residues in the oxyanion hole that stabilizes the transition state for ester hydrolysis. Furthermore, the crystal structure of the bacterial cocaine esterase (cocE) also showed that the cocE employs a tyrosine hydroxyl in the oxyanion hole. Thus, the biochemical and structural data are consistent with the catalytic antibody providing oxyanion stabilization as its major contribution to catalysis.     

The ionic track in the F1-ATPase from the thermophilic Bacillus PS3

Only beta-beta cross-links form when the alpha(3)(betaE(395)C)(3)gammaK(36)C (MF(1) residue numbers) double mutant subcomplex of TF(1), the F(1)-ATPase from the thermophilic Bacillus PS3, is slowly inactivated with CuCl(2) in the presence or absence of MgATP. The same slow rate of inactivation and extent of beta-beta cross-linking occur upon treatment of the alpha(3)(betaE(395)C)(3)gamma single mutant subcomplex with CuCl(2) under the same conditions. In contrast, the alpha(3)(betaE(395)C)(3)gammaR(33)C and alpha(3)(betaE(395)C)(3)gammaR(75)C double mutant subcomplexes of TF(1) are rapidly inactivated by CuCl(2) under the same conditions that is accompanied by complete beta-gamma cross-linking. The ATPase activity of each mutant enzyme containing the betaE(395)C substitution is stimulated to a much greater extent by the nonionic detergent lauryldimethylamine oxide (LDAO) than wild-type enzyme, whereas the ATPase activities of the gammaR(33)C, gammaK(36)C, and gammaR(75)C single mutants are stimulated to about the same extent as wild-type enzyme by LDAO. This indicates that the E(395)C substitution in the (394)DELSEED(400) segment of beta subunits increases propensity of the enzyme to entrap inhibitory MgADP in a catalytic site during turnover. These results are discussed in perspective with (i) the ionic track predicted from molecular dynamics simulations to operate during energy-driven ATP synthesis by MF(1), the F(1)-ATPase from bovine heart mitochondria [Ma, J., Flynn, T. C., Cui, Q., Leslie, A. G. W., Walker, J. E., and Karplus, M. (2002) Structure 10, 921-931]; and (ii) the possibility that the betaE(395)C substitution might induce a global effect that alters affinity of noncatalytic sites for nucleotides or alters communication between noncatalytic sites and catalytic sites during ATP hydrolysis.     

Ruminococcus albus 8 mutants defective in cellulose degradation are deficient in two processive endocellulases, Cel48A and Cel9B, both of which possess a novel modular architecture

The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized. Subtractive enrichment procedures were used to isolate mutants of R. albus 8 that are defective in adhesion to cellulose. Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked. Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose. The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R. albus 8. One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A). Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B(2) (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively. Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex. Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus. Such X modules are also present in several other R. albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.     

Influenza hemagglutinins outside of the contact zone are necessary for fusion pore expansion

Current models for membrane fusion in diverse biological processes are focused on the local action of fusion proteins present in the contact zone where the proteins anchored in one membrane might interact directly with the other membrane. Are the fusion proteins outside of the contact zone just bystanders? Here we assess the role of these "outsider" proteins in influenza virus hemagglutinin-mediated fusion between red blood cells and either hemagglutinin-expressing cells or viral particles. To selectively inhibit or enhance the actions of hemagglutinin outsiders, the antibodies that bind to hemagglutinin and proteases that cleave it were conjugated to polystyrene microspheres too large to enter the contact zone. We also involved hemagglutinin outsiders into interactions with additional red blood cells. We find the hemagglutinin outsiders to be necessary and sufficient for fusion. Interfering with the activity of the hemagglutinin outsiders inhibited fusion. Selective conversion of hemagglutinin outsiders alone into fusion-competent conformation was sufficient to achieve fusion. The discovered functional role of fusion proteins located outside of the contact zone suggests a tempting analogy to mechanisms by which proteins mediate membrane fission from outside of the fission site.     

A novel AP-2 adaptor interaction motif initially identified in the long-splice isoform of synaptojanin 1, SJ170

Phosphoinositides play a fundamental role in clathrin-coat assembly at the cell surface. Several endocytic components and accessory factors contain independently folded phosphoinositide-binding modules that facilitate, in part, membrane placement at the bud site. As the clathrin-coat assembly process progresses toward deeply invaginated buds, focally synthesized phosphoinositides are dephosphorylated, principally through the action of the phosphoinositide polyphosphatase synaptojanin 1. Failure to catabolize polyphosphoinositides retards the fission process and endocytic activity. The long-splice isoform of synaptojanin 1, termed SJ170, contains a carboxyl-terminal extension that harbors interaction motifs for engaging several components of the endocytic machinery. Here, we demonstrate that in addition to DPF and FXDXF sequences, the SJ170 carboxyl terminus contains a novel AP-2 binding sequence, the WXXF motif. The WXXF sequence engages the independently folded alpha-subunit appendage that projects off the heterotetrameric AP-2 adaptor core. The endocytic protein kinases AAK1 and GAK also contain functional WXX(FW) motifs in addition to two DPF repeats, whereas stonin 2 harbors three tandem WXXF repeats. Each of the discrete SJ170 adaptor-interaction motifs bind to appendages relatively weakly but, as tandemly arrayed within the SJ170 extension, can cooperate to bind bivalent AP-2 with good apparent affinity. These interactions likely contribute to the appropriate targeting of certain endocytic components to clathrin bud sites assembling at the cell surface.