A retroviral vector capable of targeted gene transfer into cells expressing HIV envelope glycoprotein

An expression vector encoding a chimeric envelope protein composed of CD4 and ecotropic retroviral envelope glycoprotein was constructed with the aim of accomplishing targeted gene transfer into HIV-1-infected cells. The chimeric protein was efficiently expressed and transported to the surfaces of various cell types and supported HIV-1 entry into human cells. A packaging cell line producing retroviral vectors carrying chimeric envelope proteins was then established. The vector particles produced were shown to be capable of specific gene transfer into human cells expressing HIV envelope glycoprotein. Blocking experiments confirmed that the vector particles entered the cells via an interaction between the chimeric and HIV envelope proteins. This targeting vector may thus be a useful tool with which to develop effective gene therapies against HIV infection.     

Neutralizing single-chain antibodies against the tick-borne encephalitis virus

A recombinant pSC13D6 plasmid DNA was constructed based on cDNA fragments of genes encoding variable domains of heavy and light chains of the MKA13D6 monoclonal antibody against glycoprotein of the tick-borne encephalitis (TBE) virus. This plasmid provided expression in Escherichia coli cells of the scl3D6 single-chain antibody against the TBE virus. The produced antibodies could bind to the TBE virus, strain 205, and the TBE virus recombinant E protein. The affinity constant of purified scl3D6 was (3.0 ± 0.2) × 10⁷ M^?1 for the equilibrium state and (2.8 ± 0.3) × 10⁷ M^?1 in the case of antigen-antibody formation on the surface. The obtained single-chain antibody could inhibit the infection potency of the TBE virus on a monolayer of eukaryotic cells. The calculated IC₅₀ value for scl3D6 was 16.7 μg/ml.     

Theoretical structural insights into the snakin/GASA family

Among the main classes of cysteine-stabilized antimicrobial peptides, the snakin/GASA family has not yet had any structural characterization. Through the combination of ab initio and comparative modeling with a disulfide bond predictor, the three-dimensional structure prediction of snakin-1 is reported here. The structure was composed of two long α-helices with a disulfide pattern of Cys^I-Cys^IX, Cys^II-Cys^VII, Cys^III-Cys^IV, Cys^V-Cys^XI, Cys^VI-Cys^XII and Cys^VIII-Cys^X. The overall structure was maintained throughout molecular dynamics simulation. Snakin-1 showed a small degree of structural similarity with thionins and α-helical hairpins. This is the first report of snakin-1 structural characterization, shedding some light on the snakin/GASA family.     

Specificity of anti-peptide antibodies elicited against synthetic peptides mimicking conserved regions of HIV1 envelope glycoprotein

Comparison of HIV1_Bru and HIV2_Rod external envelope glycoprotein sequences enabled us to select ten highly conserved peptide sequences. The corresponding peptides were chemically synthesized, then coupled to bovine serum albumin before injection in rabbits. Although all peptides were immunogenic, only antibodies directed against peptides P1 (amino acid residues 33–55), P22 (418–462), P8 (487–508) and P21 (487–534) were able to interact with significant affinity (K_0.5 about 10⁻⁶ and 10⁻⁸ M) with the native glycoprotein by radioimmunoassay. Noteworthy was the capacity of anti-P1 antibodies to also recognize the glycoprotein of HIV2. Anti-peptide antibodies were tested for their ability to interfere with the gp120-CD4 interaction, membrane fusion and virus replication. Preincubation of gp 120 with antibodies directed to the region previously described as the putative CD4-binding site, P22 (418–462), did not abolish gp120 binding to CD4-positive cells.     

Improved elicitation of neutralizing antibodies against primary human immunodeficiency viruses by soluble stabilized envelope glycoprotein trimers

Human immunodeficiency virus (HIV-1) envelope glycoprotein subunits, such as the gp120 exterior glycoprotein, typically elicit antibodies that neutralize T-cell-line-adapted (TCLA), but not primary, clinical isolates of HIV-1. Here we compare the immunogenicity of gp120 and soluble stabilized trimers, which were designed to resemble the functional envelope glycoprotein oligomers of primary and TCLA HIV-1 strains. For both primary and TCLA virus proteins, soluble stabilized trimers generated neutralizing antibody responses more efficiently than gp120 did. Trimers derived from a primary isolate elicited antibodies that neutralized primary and TCLA HIV-1 strains. By contrast, trimers derived from a TCLA isolate generated antibodies that neutralized only the homologous TCLA virus. Thus, soluble stabilized envelope glycoprotein trimers derived from primary HIV-1 isolates represent defined immunogens capable of eliciting neutralizing antibodies that are active against clinically relevant HIV-1 strains.     

Structural insights into SUN-KASH complexes across the nuclear envelope

Linker of the nucleoskeleton and the cytoskeleton (LINC) complexes are composed of SUN and KASH domain-containing proteins and bridge the inner and outer membranes of the nuclear envelope. LINC complexes play critical roles in nuclear positioning, cell polarization and cellular stiffness. Previously, we reported the homotrimeric structure of human SUN2. We have now determined the crystal structure of the human SUN2-KASH complex. In the complex structure, the SUN domain homotrimer binds to three independent “hook”-like KASH peptides. The overall conformation of the SUN domain in the complex closely resembles the SUN domain in its apo state. A major conformational change involves the AA''-loop of KASH-bound SUN domain, which rearranges to form a mini β-sheet that interacts with the KASH peptide. The PPPT motif of the KASH domain fits tightly into a hydrophobic pocket on the homotrimeric interface of the SUN domain, which we termed the BI-pocket. Moreover, two adjacent protomers of the SUN domain homotrimer sandwich the KASH domain by hydrophobic interaction and hydrogen bonding. Mutations of these binding sites disrupt or reduce the association between the SUN and KASH domains in vitro. In addition, transfection of wild-type, but not mutant, SUN2 promotes cell migration in Ovcar-3 cells. These results provide a structural model of the LINC complex, which is essential for additional study of the physical and functional coupling between the cytoplasm and the nucleoplasm.     

Monoclonal antibodies prepared against the major Drosophila nuclear Matrix-pore complex-lamina glycoprotein bind specifically to the nuclear envelope in situ

A high molecular weight glycoprotein found associated with a nuclear matrix-pore complex-lamina (NMPCL) preparation obtained from Drosophila melanogaster embryos has been shown by in vitro analyses to be largely confined to this subcellular fraction. In contrast with several of the NMPCL proteins, this glycoprotein remains completely insoluble after treatment with 5 M urea. It has, therefore, been possible to separate the glycoprotein from other NMPCL components by differential urea extraction. The glycoprotein in the 5 M urea-extracted pellet has been solubilized by boiling in sodium dodecyl sulfate and purified to near-homogeneity by sequential steps of chromatography on hydroxylapatite and Sephacryl S-300 (both run in the presence of 0.1% sodium dodecyl sulfate), followed by affinity chromatography on lentil lectin-Sepharose. Over 30 hybridoma cell lines producing antibodies against this glycoprotein have been obtained. Monoclonality has been established for two of these lines (designated AGP-26 and AGP-78), and the antibodies they secrete have been further characterized. Western blot analysis has shown both antibodies to be monospecific (with respect to other Drosophila embryo polypeptides) for the major NMPCL glycoprotein; in addition, antibody AGP-78 has been shown to be weakly cross-reactive with glycoproteins of similar or identical molecular weight found associated with isolated nuclear fractions obtained from Xenopus oocytes, as well as chicken, opossum, and rat livers. Finally, both antibodies AGP-26 and AGP-78 react exclusively with the Drosophila nuclear periphery (nuclear envelope) in situ as demonstrated by indirect immunofluorescence analysis of larval cryosections. Based on these results as well as upon those of biochemical studies reported previously (Berrios, M., Filson, A. J., Blobel, G, and Fisher, P. A. (1983) J. Biol. Chem. 258, 13384-13390), we conclude that the major Drosophila NMPCL glycoprotein is the specific homolog of the high molecular weight glycoprotein recently shown using immunoelectron microscopy to be a distinct component of the rat liver nuclear pore complex (Gerace, L., Ottaviano, Y., and Kondor-Koch, C. (1982) J. Cell Biol. 95, 826-837).     

Humanization and epitope mapping of neutralizing anti-human Fas ligand monoclonal antibodies: structural insights into Fas/Fas ligand interaction.

Fas ligand (L)/CD95L, a proapoptotic member of the TNF family, is a potential target for clinical intervention in various diseases. In the present study, we generated a humanized anti-human FasL mAb and characterized the epitopes of neutralizing mAbs by extensive alanine-scanning mutagenesis of human FasL. The predicted molecular model of FasL trimer revealed that the mAbs recognize largely overlapped conformational epitopes that are composed of two clusters, one around the outer tip-forming D-E loop and another near the top of FasL. Both of these sites on FasL are critically involved in the direct interaction with the corresponding receptor, Fas. These results suggest that the mAbs efficiently neutralize FasL cytotoxicity by masking both of these FasL/Fas contact sites.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Phage peptide libraries constitute powerful tools for themapping of epitopes recognized by monoclonal antibodies (mAbs).Using screening of phage displayed random peptide libraries wehave characterized the binding epitopes of three mAbs directedagainst the surface envelope glycoprotein (gp46) of the humanT-cell leukemia virus type I (HTLV-I). Two phage libraries,displaying random heptapeptides with or without flankingcysteine residues, were screened for binding to mAbs 7G5D8, DB4and 4F5F6. The SSSSTPL consensus sequence isolated fromconstrained heptapeptide library defines the epitope recognizedby DB4 mAb and corresponds to the exact region 249–252 of thevirus sequence. The APPMLPH consensus sequence isolated fromnon constrained heptapeptide library defines the epitoperecognized by 7G5D8 mAb and corresponds to the region 187–193with a single amino acid substitution, methionine to leucine atposition 190. The third consensus sequence LYWPHD isolated fromconstrained heptapeptide library defines the epitope recognizedby 4F5F6 mAb. It corresponds to an epitope without directequivalence with the virus sequence. The data presented hereshowed that 7G5D8 and DB4 mAbs are raised against linearepitopes while 4F5F6 mAb recognized a continuous topographic epitope.     

Identification of epitopes recognized by monoclonal antibodies directed against HTLV-I envelope surface glycoprotein using peptide phage display

Summary Phage peptide libraries constitute powerful tools for the mapping of epitopes recognized by monoclonal antibodies (mAbs). Using screening of phage displayed random peptide libraries we have characterized the binding epitopes of three mAbs directed against the surface envelope glycoprotein (gp46) of the human T-cell leukemia virus type I (HTLV-I). Two phage libraries, displaying random heptapeptides with or without flanking cysteine residues, were screened for binding to mAbs 7G5D8, DB4 and 4F5F6. The SSSSTPL consensus sequence isolated from constrained heptapeptide library defines the epitope recognized by DB4 mAb and corresponds to the exact region 249–252 of the virus sequence. The APPMLPH consensus sequence isolated from non constrained heptapeptide library defines the epitope recognized by 7G5D8 mAb and corresponds to the region 187–193 with a single amino acid substitution, methionine to leucine at position 190. The third consensus sequence LYWPHD isolated from constrained heptapeptide library defines the epitope recognized by 4F5F6 mAb. It corresponds to an epitope without direct equivalence with the virus sequence. The data presented here showed that 7G5D8 and DB4 mAbs are raised against linear epitopes while 4F5F6 mAb recognized a continoous topographic epitope.     

Dissecting the fibrillin microfibril: structural insights into organization and function

Force-bearing tissues such as blood vessels, lungs, and ligaments depend on the properties of elasticity and flexibility. The 10 to 12 nm diameter fibrillin microfibrils play vital roles in maintaining the structural integrity of these highly dynamic tissues and in regulating extracellular growth factors. In humans, defective microfibril function results in several diseases affecting the skin, cardiovascular, skeletal, and ocular systems. Despite the discovery of fibrillin-1 having occurred more than two decades ago, the structure and organization of fibrillin monomers within the microfibrils are still controversial. Recent structural data have revealed strategies by which fibrillin is able to maintain its architecture in dynamic tissues without compromising its ability to?interact with itself and other cell matrix components. This review summarizes our current knowledge of microfibril structure, from individual fibrillin domains and the calcium-dependent tuning of pairwise interdomain interactions to microfibril dynamics, and how this relates to microfibril function in health and disease.     

Neutralizing chimeric mouse-human antibodies against Burkholderia pseudomallei protease: expression, purification and characterization

A recombinant Fab monoclonal antibody (Fab) C37, previously obtained by phage display and biopanning of a random antibody fragment library against Burkholderia pseudomallei protease, was expressed in different strains of Escherichia coli. E. coli strain HB2151 was deemed a more suitable host for Fab expression than other E. coli strains when grown in media supplemented with 0.2 % glycerol. The expressed Fab fragment was purified by affinity chromatography on a Protein G-Sepharose column, and the specificity of the recombinant Fab C37 towards B. pseudomallei protease was proven by Western blotting, enzyme-linked immunosorbent assay (ELISA) and by proteolytic activity neutralization. In addition, polyclonal antibodies against B. pseudomallei protease were produced in rabbits immunized with the protease. These were isolated from high titer serum by affinity chromatography on recombinant-Protein A-Sepharose. Purified polyclonal antibody specificity towards B. pseudomallei protease was proven by Western blotting and ELISA.     

The spindle checkpoint: structural insights into dynamic signalling

Chromosome segregation is a complex and astonishingly accurate process whose inner working is beginning to be understood at the molecular level. The spindle checkpoint plays a key role in ensuring the fidelity of this process. It monitors the interactions between chromosomes and microtubules, and delays mitotic progression to allow extra time to correct defects. Here, we review and integrate findings on the dynamics of checkpoint proteins at kinetochores with structural information about signalling complexes.     

Outer membrane translocons: structural insights into channel formation

Gram-negative bacteria need to maintain the integrity of their outer membrane while also regulating the secretion of toxins and other macromolecules. A variety of dedicated outer membrane proteins (OMPs) facilitate this process. Recent structural work has shown that some of these proteins adopt classical β-barrel transmembrane structures and rely on structural changes within the barrel lumen to allow passage of substrate proteins. Other secretion systems have OMP components which use transmembrane α-helices and appear to function in a different way. Here we review a selection of recent structural studies which have major ramifications for our understanding of the passage of macromolecules across the outer membrane.     

Lactoperoxidase: structural insights into the function,ligand binding and inhibition

Lactoperoxidase (LPO) is a member of a large group of mammalian heme peroxidases that include myeloperoxidase (MPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). The LPO is found in exocrine secretions including milk. It is responsible for the inactivation of a wide range of micro-organisms and hence, is an important component of defense mechanism in the body. With the help of hydrogen peroxide, it catalyzes the oxidation of halides, pseudohalides and organic aromatic molecules. Historically, LPO was isolated in 1943, nearly seventy years ago but its three-dimensional crystal structure has been elucidated only recently. This review provides various details of this protein from its discovery to understanding its structure, function and applications. In order to highlight species dependent variations in the structure and function of LPO, a detailed comparison of sequence, structure and function of LPO from various species have been made. The structural basis of ligand binding and distinctions in the modes of binding of substrates and inhibitors have been analyzed extensively.     

BRing it on: new insights into the mechanism of brassinosteroid action

Several recent breakthroughs have filled in key details of the brassinosteroid (BR) response. Identification of BAK1, a BRI1 interacting protein, the negative regulator BIN2, as well as direct targets of BIN2, BZR1 and BES1, provide a link between BR perception at the cell surface and regulation of gene expression in the nucleus. Global expression studies further defined the downstream events in this pathway, confirming the role of several factors acting in negative feedback regulation on BR levels. New links to the plant hormone, auxin, were also uncovered.