Protective effect of vaginal application of neutralizing and nonneutralizing inhibitory antibodies against vaginal SHIV challenge in macaques.

Definition of antibody (Ab) functions capable of preventing mucosal HIV transmission may be critical to both effective vaccine development and the prophylactic use of monoclonal Abs. Although direct antibody-mediated neutralization is highly effective against cell-free virus, increasing evidence suggests an important role for immunoglobulin G (IgG) Fcγ receptor (FcγR)-mediated inhibition of HIV replication. Thus, a panel of well-known neutralizing (NAbs) and nonneutralizing Abs (NoNAbs) were screened for their ability to block HIV acquisition and replication in vitro in either an independent or FcγR-dependent manner. Abs displaying the highest Fc-mediated inhibitory activity in various in vitro assays were selected, formulated for topical vaginal application in a microbicide gel, and tested for their antiviral activity against SHIVSF162P3 vaginal challenge in non-human primates (NHPs). A combination of three NAbs, 2G12, 2F5, and 4E10, fully prevented simian/human immunodeficiency virus (SHIV) vaginal transmission in 10 out of 15 treated NHPs, whereas a combination of two NoNAbs, 246-D and 4B3, although having no impact on SHIV acquisition, reduced plasma viral load. These results indicate that anti-HIV Abs with distinct neutralization and inhibitory functions differentially affect in vivo HIV acquisition and replication, by interfering with early viral replication and dissemination. Therefore, combining diverse Ab properties may potentiate the protective effects of anti-HIV-Ab-based strategies.

A reinvestigation provides no evidence for sugar residues on structural proteins of poleroviruses and argues against a role for glycosylation of virus structural proteins in aphid transmission.

Poleroviruses are strictly transmitted by aphids. Glycosylation of Turnip yellows virus (TuYV) was previously reported and this modification was supposed to be required for aphid transmission. Using different approaches based on (i) a lectin-binding assay, (ii) use of specific complex glycan antibodies and (iii) mass spectrometry, we found no evidence that the structural proteins of TuYV and Cucurbit aphid-borne yellow virus (CABYV) carry glycan residues. Moreover, mutation of each of the four potential N-glycosylation sites of the structural protein sequences of CABYV indicated that, unless more than one site on the structural protein is glycosylated, N-glycosylation is not involved in aphid transmission. These results did not corroborate the previous hypothesis for the role of glycosylation in aphid transmission. They, however, revealed the presence of a glycosylated plant protein in purified polerovirus suspensions, whose function in aphid transmission should be further investigated.

Native MS: an 'ESI' way to support structure- and fragment-based drug discovery.

The success of early drug-discovery programs depends on the adequate combination of complementary and orthogonal technologies allowing hit/lead compounds to be optimized and improve therapeutic activity. Among the available biophysical methods, native MS recently emerged as an efficient method for compound-binding screening. Native MS is a highly sensitive and accurate screening technique. This review provides a description of the general approach and an overview of the possible characterization of ligand-binding properties. How native MS supports structure- and fragment-based drug research will also be discussed, with examples from the literature and internal developments. Native MS shows strong potential for in-depth characterization of ligand-binding properties. It is also a reliable screening technique in drug-discovery processes.

Analytical identification of additional impurities in urinary-derived gonadotrophins.

Advances in proteomic technology have enabled contaminant proteins to be identified from complex protein mixtures. The purity of two purified urinary gonadotrophin products, human menopausal gonadotrophin (u-HMG) and human FSH (u-hFSH), was compared with a preparation of recombinant human FSH (r-hFSH). After separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), western blot analysis showed that the recombinant preparation contained only FSH, whereas the urine-derived preparations exhibited several non-FSH or LH-related bands. These urinary components were further investigated by a proteomic approach using two-dimensional SDS-PAGE followed by mass spectrometric identification. The proteomic approach detected a total of 23 non-gonadotrophin-related proteins, at variable levels in different batches of the urine-derived preparations. Of these, 16 co-purified proteins have not been previously reported to be present in urine-derived gonadotrophins. These results indicate that the process used to purify urinary gonadotrophins may not remove all non-gonadotrophin proteins. By using a comprehensive proteomic approach, it has been shown that the recombinant FSH preparation has greater purity than either of the urine-derived preparations.

Chfr interacts and colocalizes with TCTP to the mitotic spindle.

Chfr is a checkpoint protein that plays an important function in cell cycle progression and tumor suppression, although its exact role and regulation are unclear. Previous studies have utilized overexpression of Chfr to determine the signaling pathway of this protein in vivo. In this study, we demonstrate, by using three different antibodies against Chfr, that the endogenous and highly overexpressed ectopic Chfr protein is localized and regulated differently in cells. Endogenous and lowly expressed ectopic Chfr are cytoplasmic and localize to the spindle during mitosis. Higher expression of ectopic Chfr correlates with a shift in the localization of this protein to the nucleus/PML bodies, and with a block of cell proliferation. In addition, endogenous and lowly expressed ectopic Chfr is stable throughout the cell cycle, whereas when highly expressed, ectopic Chfr is actively degraded during S-G2/M phases in an autoubiquitination and proteasome-dependent manner. A two-hybrid screen identified TCTP as a possible Chfr-interacting partner. Biochemical analysis with the endogenous proteins confirmed this interaction and identified beta-tubulin as an additional partner for Chfr, supporting the mitotic spindle localization of Chfr. The Chfr-TCTP interaction was stable throughout the cell cycle, but it could be diminished by the complete depolymerization of the microtubules, providing a possible mechanism where Chfr could be the sensor that detects microtubule disruption and then activates the prophase checkpoint.

[Discovery and crystallographic structure of human apolipoprotein]. / Découverte et structure cristallographique d'une apolipoprotéine humaine.

We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is co-purified with paraoxonase (PON1). The association between HPON1 and HPBP is modulated by phosphate and calcium concentrations. The HPBP X-ray structure solved at 1.9 A resolution is similar to the prokaryotic phosphate solute-binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation of genes between evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the first identified transporter capable of binding phosphate ions in human plasma. Thus it is thought to become a new predictor and a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.

High-resolution neutron protein crystallography with radically small crystal volumes: application of perdeuteration to human aldose reductase.

Neutron diffraction data have been collected to 2.2 Angstrom resolution from a small (0.15 mm(3)) crystal of perdeuterated human aldose reductase (h-AR; MW = 36 kDa) in order to help to determine the protonation state of the enzyme. h-AR belongs to the aldo-keto reductase family and is implicated in diabetic complications. Its ternary complexes (h-AR-coenzyme NADPH-selected inhibitor) provide a good model to study both the enzymatic mechanism and inhibition. Here, the successful production of fully deuterated human aldose reductase [h-AR(D)], subsequent crystallization of the ternary complex h-AR(D)-NADPH-IDD594 and neutron Laue data collection at the LADI instrument at ILL using a crystal volume of just 0.15 mm(3) are reported. Neutron data were recorded to 2 Angstrom resolution, with subsequent data analysis using data to 2.2 Angstrom. This is the first fully deuterated enzyme of this size (36 kDa) to be solved by neutron diffraction and represents a milestone in the field, as the crystal volume is at least one order of magnitude smaller than those usually required for other high-resolution neutron structures determined to date. This illustrates the significant increase in the signal-to-noise ratio of data collected from perdeuterated crystals and demonstrates that good-quality neutron data can now be collected from more typical protein crystal volumes. Indeed, the signal-to-noise ratio is then dominated by other sources of instrument background, the nature of which is under investigation. This is important for the design of future instruments, which should take maximum advantage of the reduction in the intrinsic diffraction pattern background from fully deuterated samples.

Study on agglutinating factors from flocculent Saccharomyces cerevisiae strains.

The lectin-like theory suggest that yeast flocculation is mediated by an aggregating lectinic factor. In this study we isolated an agglutinating factor, which corresponds to lectin, from whole cells by treating the flocculent wild-type Saccharomyces cerevisiae NCYC 625 strain and its weakly flocculent mutant [rho degrees ] with EDTA and two non-ionic surfactants (Hecameg and HTAC). The dialysed crude extracts obtained in this way agglutinated erythrocytes and this hemagglutination was specifically inhibited by mannose and mannose derivatives. However, SDS-PAGE profiles showed that the three reagents had different effects on the yeast cells. The non-ionic surfactants appeared to be the most efficient, as their extracts possessed the highest specific agglutinating activity. The products released by the wild-type strain presented a higher specific agglutinating activity than those released by the [rho degrees ] mutant. Purification of the agglutinating factor from extracts of both strains by affinity chromatography revealed two active bands of relative mass of 26 and 47 kDa on SDS-PAGE. Mass spectrometry analysis by MALDI-TOF, identified a 26 kDa band as the triose phosphate isomerase (TPI) whereas a 47 kDa band was identical to enolase. Edman degradation showed that the N-terminal sequences of these proteins were similar to TPI and enolase, respectively. The difference in the flocculation behaviour of the two strains is due to changes in the protein composition of the cell wall and in the protein structure involved in cell-cell recognition.

Expression, purification, crystallization and preliminary X-ray analysis of the cathelicidin motif of the protegrin-3 precursor.

Numerous precursors of antibacterial peptides with unrelated sequences share a similar prosequence which belongs to the cathelicidin family of proteins. The three-dimensional structure of this cathelicidin motif, which contains two disulfide bonds, has not yet been reported. The cathelicidin motif (ProS) of the protegrin-3 precursor was overexpressed in Escherichia coli as a His-tagged protein. The His(6) tag was removed by thrombin cleavage. ProS was purified to homogeneity and single crystals were obtained by the hanging-drop vapour-diffusion method at pH 3-4. Preliminary X-ray diffraction analysis indicated that these crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 51.42, c = 134.25 A. These crystals diffracted beyond 2.75 A (1.9 A at ESRF) and contain one molecule per asymmetric unit.

A comparison between Sypro Ruby and ruthenium II tris (bathophenanthroline disulfonate) as fluorescent stains for protein detection in gels.

A comparison between two fluorescent metal chelates for staining proteins separated by electrophoresis has been carried out. One of these chelates is ruthenium II tris (bathophenanthroline disulfonate) and the other is commercial Sypro Ruby. Both can be efficiently detected either with UV tables or with commercial laser fluorescence scanners. The sensitivity and homogeneity of the stains and the interference with mass spectrometry analysis have been investigated. It appears that both stains perform similarly for protein detection, while ruthenium II tris (bathophenanthroline disulfonate) performs better for mass spectrometry analyses and as cost-effectiveness ratio. However, Sypro Ruby is easier to use as a stain.

The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p.

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430-650-kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430-650-kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate for the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact.

Cost-effective and uniform (13)C- and (15)N-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp. PCC 7120.

Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes. While (13)C/(15)N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp. PCC 7120 can be uniformly labeled by growing them in medium containing Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-labeled proteins in Anabaena sp. PCC 7120. The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of (13)C and (15)N enrichment in the constitutively expressed 24-kDa polypeptide. The yield of purified 24-kDa protein after dual isotope labeling under anaerobic conditions was similar to that obtained with E. coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium. Furthermore, as probed by NMR spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E. coli sample, demonstrating that it was of sufficient quality for 3D-structure determination. Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp. PCC 7120 is an economic alternative for the (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies.

Identification by mass spectrometry of two-dimensional gel electrophoresis-separated proteins extracted from lager brewing yeast.

As two-dimensional (2-D) electrophoresis allows the separation of several hundred proteins in a single gel, this technique has become an important tool for proteome studies and for investigating the cellular physiology. In order to take advantage of information provided by the comparison of proteome pictures, the mass spectrometry technique is the way chosen for a rapid and an accurate identification of proteins of interest. Unfortunately, in the case of industrial yeasts, due to the high level of complexity of their genome, the whole DNA sequence is not yet available and all encoded protein sequences are still unknown. Nevertheless, this study presents here 30 lager brewing yeast proteins newly identified with matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), tandem mass spectrometry (MS/MS) and database searching against the protein sequences of Saccharomyces cerevisiae. The identified proteins of the industrial strain correspond to proteins which do not comigrate with known proteins of S. cerevisiae separated on 2-D gels. This study presents an application of the MS technique for the identification of industrial yeast proteins which are only homologous to the corresponding S. cerevisiae proteins.

Biochemical characterization of the helper component of Cauliflower mosaic virus.

The helper component of Cauliflower mosaic virus is encoded by viral gene II. This protein (P2) is dispensable for virus replication but required for aphid transmission. The purification of P2 has never been reported, and hence its biochemical properties are largely unknown. We produced the P2 protein via a recombinant baculovirus with a His tag fused at the N terminus. The fusion protein was purified by affinity chromatography in a soluble and biologically active form. Matrix-assisted laser desorption time-of-flight mass spectrometry demonstrated that P2 is not posttranslationally modified. UV circular dichroism revealed the secondary structure of P2 to be 23% alpha-helical. Most alpha-helices are suggested to be located in the C-terminal domain. Using size exclusion chromatography and aphid transmission testing, we established that the active form of P2 assembles as a huge soluble oligomer containing 200 to 300 subunits. We further showed that P2 can also polymerize as long paracrystalline filaments. We mapped P2 domains involved in P2 self-interaction, presumably through coiled-coil structures, one of which is proposed to form a parallel trimer. These regions have previously been reported to also interact with viral P3, another protein involved in aphid transmission. Possible interference between the two types of interaction is discussed with regard to the biological activity of P2.

Synthesis, mechanism of action, and antiviral activity of a new series of covalent mechanism-based inhibitors of S-adenosyl-L-homocysteine hydrolase.

A direct method for the preparation of 5'-S-alkynyl-5'-thioadenosine and 5'-S-allenyl-5'-thioadenosine has been developed. Treatment of a protected 5'-acetylthio-5'-deoxyadenosine with sodium methoxide and propargyl bromide followed by deprotection gave the 5'-S-propargyl-5'-thioadenosine 4. Under controlled base-catalysis with sodium tert-butoxide in tert-butyl alcohol 4 was quantitatively converted into 5'-S-allenyl-5'-thioadenosine 5 or 5'-S-propynyl-5'-thioadenosine 6. Incubation of recombinant human placental AdoHcy hydrolase with 4, 5, or 6 resulted in time- and concentration-dependent inactivation of the enzyme (K(i): 45 +/- 0.5, 16 +/- 1, and 15 +/- 1 microM, respectively). Compound 4 caused complete conversion of the enzyme from its E-NAD(+) to E-NADH form during the inactivation process. This indicates that 4 is a substrate for the 3'-oxidative activity of AdoHcy hydrolase (type I inhibitor). In contrast, the NAD(+)/NADH content of the enzyme was not affected during the inactivation process with 5 and 6, and their mechanism of inactivation was further investigated. Addition of enzyme-sequestered water on the S-allenylthio group of 5 or S-propynylthio group of 6 within the active site should lead to the formation of the corresponding thioester 7. This acylating-intermediate agent could then undergo nucleophilic attack by a protein residue, leading to a type II mechanism-based inactivation. ElectroSpray mass spectra analysis of the inactivated protein by 5 supports this mechanistic proposal. Further studies (MALDI-TOF and ESI/MS(n) experiments) of the trypsin and endo-Lys-C proteolytic cleavage of the fragments of inactivated AdoHcy hydrolase by 5 were carried out for localization of the labeling. The antiviral activity of 4, 5, and 6 against a large variety of viruses was determined. Significant activity (EC(50): 1.9 microM) was noted with 5 against vaccinia virus.

High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies.

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.

Mass spectrometry as a novel approach to probe cooperativity in multimeric enzymatic systems.

Investigating cooperativity in multimeric enzymes is of utmost interest to improve our understanding of the mechanism of enzymatic regulation. In the present article, we propose a novel approach based on mass spectrometry to probe cooperativity in the binding of a ligand to a multisubunit enzyme. This approach presents the selective advantage of giving a direct insight into all the subsequent ligation states that are formed in solution as the ligand is added to the enzyme. A quantitative interpretation of the electrospray ionization (ESI) mass spectra gives the relative abundance of all the distinct enzymatic species, which allows one to directly deduce the cooperativity of the system. The overall method is described for the addition of the oxidized cofactor nicotinamide adenine dinucleotide (NAD(+)) to a dimeric mutant of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase (GPDH). It is then applied to four tetrameric enzymes: sturgeon muscle GPDH, wild type and S48G mutant of GPDH from B. stearothermophilus, and alcohol dehydrogenase (ADH) from Bakers yeast. The results illustrate the possibilities offered by this new technique. First, mass spectrometry allows a control of the enzymes before the addition of NAD(+). Second, the cooperative behavior can be drawn from one single ESI mass spectrum, which makes the method highly attractive in terms of the amount of biological material required. Above all, the major benefit lies in the direct visualization of all the enzymatic species that are in equilibrium in solution. The direct measurement of cooperativity readily resolve the inconvenience of the classical approaches employed in this field, which all need to model the experimental data in order to get the cooperative behavior of the system.

Characterization and quality control of recombinant adenovirus vectors for gene therapy.

Highly purified recombinant adenovirus undergoes routine quality controls for identity, potency and purity prior to its use as a gene therapy vector. Quantitative characterization of infectivity is measurable by the expression of the DNA binding protein, an early adenoviral protein, in an immunofluorescence bioassay on permissive cells as a potency determinant. The specific particle count, a key quality indicator, is the total number of intact particles present compared to the number of infectious units. Electron microscopic analysis using negative staining gives a qualitative biophysical analysis of the particles eluted from anion-exchange HPLC. One purity assessment is accomplished via the documented presence and relative ratios of component adenoviral proteins as well as potential contaminants by reversed-phase HPLC of the intact virus followed by protein peak identification using MALDI-TOF mass spectrometry and subsequent data mining. Verification of the viral genome is performed and expression of the transgene is evaluated in in vitro systems for identity. Production lots are also evaluated for replication-competent adenovirus prior to human use. For adenovirus carrying the human IL-2 transgene, quantitative IL-2 expression is demonstrated by ELISA and cytokine potency by cytotoxic T lymphocyte assay following infection of permissive cells. Both quantitative and qualitative analyses show good batch to batch reproducibility under routine test conditions using validated methods.