Molecular and Physicochemical Factors Governing Solubility of the HIV gp41 Ectodomain.

The HIV gp41 ectodomain (e-gp41) is an attractive target for the development of vaccines and drugs against HIV because of its crucial role in viral fusion to the host cell. However, because of the high insolubility of e-gp41, most biophysical and structural analyses have relied on the production of truncated versions removing the loop region of gp41 or the utilization of nonphysiological solubilizing conditions. The loop region of gp41 is also known as principal immunodominant domain (PID) because of its high immunogenicity, and it is essential for gp41-mediated HIV fusion. In this study we identify the aggregation-prone regions of the amino acid sequence of the PID and engineer a highly soluble mutant that preserves the trimeric structure of the wild-type e-gp41 under physiological pH. Furthermore, using a reverse mutagenesis approach, we analyze the role of mutated amino acids upon the physicochemical factors that govern solubility of e-gp41. On this basis, we propose a molecular model for e-gp41 self-association, which can guide the production of soluble e-gp41 mutants for future biophysical analyses and biotechnological applications.

Absolute and multiplex quantification of antibodies in serum using PSAQ™ standards and LC-MS/MS.

BACKGROUND: In preclinical studies, monoclonal antibodies (mAbs) are traditionally assayed by ligand-binding-assays. Recently, quantitative liquid chromatography mass spectrometry (MS)-based assays have emerged which circumvent a number of challenges. These assays may also be multiplex, making them potentially compatible with pharmacokinetic assays for combined antibody therapies. MATERIALS & METHODS: We combined a quantitative MS-based approach with the protein standard for absolute quantification (PSAQ™) strategy to simultaneously quantify three mAb variants presenting minor sequence differences. Stable isotopically labeled mAbs were produced and used as quantification standards. Titration curves were performed to assess the analytical performances of the method. LC-MS/MS and ELISA data were cross-compared. RESULTS: The approach presented provides similar accuracy and precision than ELISA, while being multiplex and faster to develop. It has applications at all stages of the pharmaceutical development.

DIGESTIF: a universal quality standard for the control of bottom-up proteomics experiments.

In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions. Here, we describe the design, synthesis, and validation of a universal protein standard, called DIGESTIF, that can be added to any biological sample. The DIGESTIF standard consists of a soluble recombinant protein scaffold to which a set of 11 artificial peptides (iRT peptides) with good ionization properties has been incorporated. In the protein scaffold, the amino acids flanking iRT peptide cleavage sites were selected either to favor or hinder protease cleavage. After sample processing, the retention time and relative intensity pattern of the released iRT peptides can be used to assess the quality of sample workup, the extent of digestion, and the performance of the LC-MS system. Thus, DIGESTIF can be used to standardize a broad spectrum of applications, ranging from simple replicate measurements to large-scale biomarker screening in biomedical applications.

Single-chain protein mimetics of the N-terminal heptad-repeat region of gp41 with potential as anti-HIV-1 drugs.

During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates.

Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity.

The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 ± 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 ± 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 ± 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 ± 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the importance of this domain.

The conserved Candida albicans CA3427 gene product defines a new family of proteins exhibiting the generic periplasmic binding protein structural fold.

Nosocomial diseases due to Candida albicans infections are in constant rise in hospitals, where they cause serious complications to already fragile intensive care patients. Antifungal drug resistance is fast becoming a serious issue due to the emergence of strains resistant to currently available antifungal agents. Thus the urgency to identify new potential protein targets, the function and structure of which may guide the development of new antifungal drugs. In this context, we initiated a comparative genomics study in search of promising protein coding genes among the most conserved ones in reference fungal genomes. The CA3427 gene was selected on the basis of its presence among pathogenic fungi contrasting with its absence in the non pathogenic Saccharomyces cerevisiae. We report the crystal 3D-structure of the Candida albicans CA3427 protein at 2.1 Å resolution. The combined analysis of its sequence and structure reveals a structural fold originally associated with periplasmic binding proteins. The CA3427 structure highlights a binding site located between the two protein domains, corresponding to a sequence segment conserved among fungi. Two crystal forms of CA3427 were found, suggesting that the presence or absence of a ligand at the proposed binding site might trigger a "Venus flytrap" motion, coupled to the previously described activity of bacterial periplasmic binding proteins. The conserved binding site defines a new subfamily of periplasmic binding proteins also found in many bacteria of the bacteroidetes division, in a choanoflagellate (a free-living unicellular and colonial flagellate eukaryote) and in a placozoan (the closest multicellular relative of animals). A phylogenetic analysis suggests that this gene family originated in bacteria before its horizontal transfer to an ancestral eukaryote prior to the radiation of fungi. It was then lost by the Saccharomycetales which include Saccharomyces cerevisiae.

Immunization with HIV-1 gp41 subunit virosomes induces mucosal antibodies protecting nonhuman primates against vaginal SHIV challenges.

Human immunodeficiency virus (HIV)-1 is mainly transmitted mucosally during sexual intercourse. We therefore evaluated the protective efficacy of a vaccine active at mucosal sites. Macaca mulatta monkeys were immunized via both the intramuscular and intranasal routes with an HIV-1 vaccine made of gp41-subunit antigens grafted on virosomes, a safe delivery carrier approved in humans with self-adjuvant properties. Six months after 13 vaginal challenges with simian-HIV (SHIV)-SF162P3, four out of five vaccinated animals remained virus-negative, and the fifth was only transiently infected. None of the five animals seroconverted to p27gag-SIV. In contrast, all 6 placebo-vaccinated animals became infected and seroconverted. All protected animals showed gp41-specific vaginal IgAs with HIV-1 transcytosis-blocking properties and vaginal IgGs with neutralizing and/or antibody-dependent cellular-cytotoxicity activities. In contrast, plasma IgGs totally lacked virus-neutralizing activity. The protection observed challenges the paradigm whereby circulating antiviral antibodies are required for protection against HIV-1 infection and may serve in designing a human vaccine against HIV-1-AIDS.

Recombinant fusion proteins assembling Der p 1 and Der p 2 allergens from Dermatophagoides pteronyssinus.

BACKGROUND: Fusion proteins assembling multiple allergens can be engineered by recombinant DNA technologies in order to produce tools for diagnostic and immunotherapeutic purposes. Herein, we developed and characterized chimeras assembling Der p 1 and Der p 2 allergens as potential candidate vaccines against house dust mite allergy. METHODS: Fusion proteins encompassing Der p 2 with either mature or proDer p 1 were expressed in Escherichia coli or Pichia pastoris. Forms with mutation in Der p 1 catalytic site were also engineered. Purified chimeras were characterized by immunoblotting, circular dichroism, disulfide bond mapping, basophil and T lymphocyte stimulation assays. RESULTS: Four fusion proteins were expressed in E. coli as inclusion bodies, whereas only chimeras comprising proDer p 1 were obtained in yeast. All such hybrids formed polymers and aggregates, and yeast-expressed chimeras were unstable. Circular dichroism analysis performed after refolding of bacteria expressed chimeras encompassing mature Der p 1 confirmed partial folding, consistent with the occurrence of both correct and inappropriate intramolecular disulfide bonds. All fusion molecules were recognized by Der p 1- and Der p 2-specific human IgEs, monoclonal and polyclonal antibodies. Fusion proteins activate basophils from mite-allergic patients and trigger the proliferation of specific CD4+ T cells, albeit to a lower level when compared to individual allergens. CONCLUSIONS: Production of multiple Der p 1-Der p 2 fusion proteins exhibiting partial folding and proper antigenic properties has been achieved. Nonetheless, significant solubility and stability issues currently limit the application of such chimeras for immunotherapy or diagnostic.

Structural characterization of CA1462, the Candida albicans thiamine pyrophosphokinase.

BACKGROUND: In search of new antifungal targets of potential interest for pharmaceutical companies, we initiated a comparative genomics study to identify the most promising protein-coding genes in fungal genomes. One criterion was the protein sequence conservation between reference pathogenic genomes. A second criterion was that the corresponding gene in Saccharomyces cerevisiae should be essential. Since thiamine pyrophosphate is an essential product involved in a variety of metabolic pathways, proteins responsible for its production satisfied these two criteria. RESULTS: We report the enzymatic characterization and the crystallographic structure of the Candida albicans Thiamine pyrophosphokinase. The protein was co-crystallized with thiamine or thiamine-PNP. CONCLUSION: The presence of an inorganic phosphate in the crystallographic structure opposite the known AMP binding site relative to the thiamine moiety suggests that a second AMP molecule could be accommodated in the C. albicans structure. Together with the crystallographic structures of the enzyme/substrate complexes this suggests the existence of a secondary, less specific, nucleotide binding site in the Candida albicans thiamine pyrophosphokinase which could transiently serve during the release or the binding of ATP. The structures also highlight a conserved Glutamine residue (Q138) which could interact with the ATP alpha-phosphate and act as gatekeeper. Finally, the TPK/Thiamine-PNP complex is consistent with a one step mechanism of pyrophosphorylation.

Purification and biochemical characterization of the LIP2 lipase from Yarrowia lipolytica.

The LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) was obtained from two genetically modified strains with multi-copies of the lip2 gene and further purified using gel filtration and cation exchange chromatography. Four YLLIP2 isoforms were identified and subjected to N-terminal amino-acid sequencing and mass spectrometry analysis. These isoforms differed in their glycosylation patterns and their molecular masses ranged from 36,874 to 38,481 Da, whereas the polypeptide mass was 33,385 Da. YLLIP2 substrate specificity was investigated using short (tributyrin), medium (trioctanoin) and long (olive oil) chain triglyceride substrates at various pH and bile salt concentrations, and compared with those of human gastric and pancreatic lipases. YLLIP2 was not inhibited by bile salts at micellar concentrations with any of the substrates tested, and maximum specific activities were found to be 10,760+/-115 U/mg on tributyrin, 16,920+/-480 U/mg on trioctanoin and 12,260+/-700 U/mg on olive oil at pH 6.0. YLLIP2 was found to be fairly stable and still active on long chain triglycerides (1590+/-430 U/mg) at pH 4.0, in the presence of bile salts. It is therefore a good candidate for use in enzyme replacement therapy as a means of treating pancreatic exocrine insufficiency.

Expression optimization and purification process development of an engineered soluble recombinant mouse linker of activation of T cells using surface enhanced laser desorption/ionization-mass spectrometry.

Protein purification development is the bottleneck of recombinant protein production therefore there is a need to shorten process development and monitoring. Surface enhanced laser desorption/ionization-mass spectrometry (SELDI-MS) was evaluated to optimize the expression and to develop the purification of a recombinant mouse protein: a transmembrane adaptor involved in T cell receptor signaling named "linker for activation of T cells" (LAT). The protein was expressed as a soluble form (S-LAT) in three strains of Escherichia coli: BL21 (DE3), Rosetta (DE3), and BL21 (DE3) pLys S. The expression of S-LAT was monitored on immobilized metal affinity chromatography (IMAC) ProteinChip arrays. The highest level of expression was found in Rosetta (DE3) with a C-terminal construct after induction at 37 degrees C. The purification scheme was elucidated using SELDI-MS: S-LAT was efficiently captured on an IMAC ProteinChip array saturated with nickel ions (Ni(2+)) and then fractionated on a Q ProteinChip array. These conditions were directly transferred to IMAC-Ni(2+) HyperCel and Q Ceramic HyperD F chromatography sorbents. After these two purification steps, S-LAT was estimated to be more than 80% pure, confirming a very good match between array and sorbent. Finally, a peptide mapping was performed on a hydrophobic array after in gel trypsin digest, verifying that the purified protein was the mouse LAT. This is the first report of a protocol for the production and purification of S-LAT. The selection of the best expression and purification strategy along with the identification were enabled in 5 days with less than 5 mL of soluble fraction of crude culture samples.

Increase of the deacylation rate of PBP2x from Streptococcus pneumoniae by single point mutations mimicking the class A beta-lactamases.

The class A beta-lactamases and the transpeptidase domain of the penicillin-binding proteins (PBPs) share the same topology and conserved active-site residues. They both react with beta-lactams to form acylenzymes. The stability of the PBP acylenzymes results in the inhibition of the transpeptidase function and the antibiotic activity of the beta-lactams. In contrast, the deacylation of the beta-lactamases is extremely fast, resulting in a high turnover of beta-lactam hydrolysis, which confers resistance to these antibiotics. In TEM-1 beta-lactamase from Escherichia coli, Glu166 is required for the fast deacylation and occupies the same spatial location as Phe450 in PBP2x from Streptococcus pneumoniae. To gain insight into the deacylation mechanism of both enzymes, Phe450 of PBP2x was replaced by various residues. The introduction of ionizable side chains increased the deacylation rate, in a pH-dependent manner, for the acidic residues. The aspartic acid-containing variant had a 110-fold faster deacylation at pH 8. The magnitude of this effect is similar to that observed in a naturally occurring variant of PBP2x, which confers increased resistance to cephalosporins.