Identification of PDE6D as a molecular target of anecortave acetate via a methotrexate-anchored yeast three-hybrid screen.

Glaucoma and age-related macular degeneration are ocular diseases targeted clinically by anecortave acetate (AA). AA and its deacetylated metabolite, anecortave desacetate (AdesA), are intraocular pressure (IOP)-lowering and angiostatic cortisenes devoid of glucocorticoid activity but with an unknown mechanism of action. We used a methotrexate-anchored yeast three-hybrid (Y3H) technology to search for binding targets for AA in human trabecular meshwork (TM) cells, the target cell type that controls IOP, a major risk factor in glaucoma. Y3H hits were filtered by competitive Y3H screens and coimmunoprecipitation experiments and verified by surface plasmon resonance analysis to yield a single target, phosphodiesterase 6-delta (PDE6D). PDE6D is a prenyl-binding protein with additional function outside the PDE6 phototransduction system. Overexpression of PDE6D in mouse eyes caused elevated IOP, and this elevation was reversed by topical ocular application of either AA or AdesA. The identification of PDE6D as the molecular binding partner of AA provides insight into the role of this drug candidate in treating glaucoma.

Yeast "N"-hybrid systems for protein-protein and drug-protein interaction discovery.

The majority of small molecule drugs act on protein targets to exert a therapeutic function. It has become apparent in recent years that many small molecule drugs act on more than one particular target and consequently, approaches which profile drugs to uncover their target binding spectrum have become increasingly important. Classical yeast two-hybrid systems have mainly been used to discover and characterize protein-protein interactions, but recent modifications and improvements have opened up new routes towards screening for small molecule-protein interactions. Such yeast "n"-hybrid systems hold great promise for the development of drugs which interfere with protein-protein interactions and for the discovery of drug-target interactions. In this review, we discuss several yeast two-hybrid based approaches with applications in drug discovery and describe a protocol for yeast three-hybrid screening of small molecules to identify their direct targets.

Functional analyses of mycobacterial lipoprotein diacylglyceryl transferase and comparative secretome analysis of a mycobacterial lgt mutant.

Preprolipopoprotein diacylglyceryl transferase (Lgt) is the gating enzyme of lipoprotein biosynthesis, and it attaches a lipid structure to the N-terminal part of preprolipoproteins. Using Lgt from Escherichia coli in a BLASTp search, we identified the corresponding Lgt homologue in Mycobacterium tuberculosis and two homologous (MSMEG_3222 and MSMEG_5408) Lgt in Mycobacterium smegmatis. M. tuberculosis lgt was shown to be essential, but an M. smegmatis ΔMSMEG_3222 mutant could be generated. Using Triton X-114 phase separation and [(14)C]palmitic acid incorporation, we demonstrate that MSMEG_3222 is the major Lgt in M. smegmatis. Recombinant M. tuberculosis lipoproteins Mpt83 and LppX are shown to be localized in the cell envelope of parental M. smegmatis but were absent from the cell membrane and cell wall in the M. smegmatis ΔMSMEG_3222 strain. In a proteomic study, 106 proteins were identified and quantified in the secretome of wild-type M. smegmatis, including 20 lipoproteins. All lipoproteins were secreted at higher levels in the ΔMSMEG_3222 mutant. We identify the major Lgt in M. smegmatis, show that lipoproteins lacking the lipid anchor are secreted into the culture filtrate, and demonstrate that M. tuberculosis lgt is essential and thus a validated drug target.

Regulation of ENaC biogenesis by the stress response protein SERP1.

Cystic fibrosis lung disease is caused by reduced Cl(-) secretion along with enhanced Na(+) absorption, leading to reduced airway surface liquid and compromised mucociliary clearance. Therapeutic strategies have been developed to activate cystic fibrosis transmembrane conductance regulator (CFTR) or to overcome enhanced Na(+) absorption by the epithelial Na(+) channel (ENaC). In a split-ubiquitin-based two-hybrid screening, we identified stress-associated ER protein 1 (SERP1)/ribosome-associated membrane protein 4 as a novel interacting partner for the ENaC ß-subunit. SERP1 is induced during cell stress and interacts with the molecular chaperone calnexin, thus controlling early biogenesis of membrane proteins. ENaC activity was measured in the human airway epithelial cell lines H441 and A549 and in voltage clamp experiments with ENaC-overexpressing Xenopus oocytes. We found that expression of SERP1 strongly inhibits amiloride-sensitive Na(+) transport. SERP1 coimmunoprecipitated and colocalized with ßENaC in the endoplasmic reticulum, together with the chaperone calnexin. In contrast to the inhibitory effects on ENaC, SERP1 appears to promote expression of CFTR. Taken together, SERP1 is a novel cochaperone and regulator of ENaC expression.

Application of the split-protein sensor Trp1 to protein interaction discovery in the yeast Saccharomyces cerevisiae.

Yeast two-hybrid based systems are powerful tools for the detection and characterization of protein-protein interactions (PPIs). However, some important protein classes, e.g., integral membrane proteins and transcription factors, are difficult to study using these technologies. To overcome these limitations, we have employed a novel protein complementation screening platform. Protein interactions are detected by reconstitution of the split-protein sensor TRP1, enabling trp1 cells to grow on medium lacking tryptophan. Since the interaction readout is direct and independent of transcriptional reporter activation the rate of false positives is lowered. Furthermore, the technology allows for detection of protein interactions in their natural setting, e.g., the cytosol, the nucleus, and at cellular or organellar membranes. The protocols used for screening are explained in detail and as an example we describe the isolation of novel binding partners found with APP screened against a human cDNA library.

Lipoprotein synthesis in mycobacteria.

Lipoproteins are a functionally diverse class of secreted bacterial proteins characterized by an N-terminal lipid moiety. The lipid moiety serves to anchor these proteins to the cell surface. Lipoproteins are synthesized as pre-prolipoproteins and mature by post-translational modifications. The post-translational modifications are directed by the lipobox motif located within the signal peptide. Enzymes involved in lipoprotein synthesis are essential in Gram-negative bacteria but not in Gram-positive bacteria. Inactivation of genes involved in lipoprotein synthesis attenuates a variety of Gram-positive pathogens, including Mycobacterium tuberculosis. The attenuated phenotype of these mutants indicates an important role of lipoproteins and lipoprotein synthesis in bacterial virulence. M. tuberculosis, the causative agent of tuberculosis, is one of the most devastating pathogens in the world. This article reviews recent findings on the synthesis, localization and function of lipoproteins in mycobacteria.

Breaking down the wall: fractionation of mycobacteria.

Mycobacterium spp. possess a complex cell envelope that consists of a plasma membrane, a peptidoglycan-arabinogalactan complex which in turn is esterified by mycolic acids that form with other non-bound lipids an asymmetric permeability barrier and an outer layer, also called a capsule in the case of pathogenic species. In order to investigate the functional roles of the cell envelope components, especially those of the major pathogens Mycobacterium tuberculosis and Mycobacterium leprae, it is necessary to fractionate the envelope by breaking the unusual wall that covers these bacteria. To this aim we first compared the efficiency of high pressure (cell disrupter/French press) with those of pathogen-compatible breakage methods such as sonication, bead beater and lysozyme treatment using the non-pathogenic Mycobacterium smegmatis. When the distribution of various specific markers of the cell envelope compartments, which include mycolic acids, arabinose, NADH oxidase activity, cell wall and cytosolic proteins, were determined sonication combined with lysozyme treatment was found to be the best option. The protocol of subcellular fractionation was then validated for pathogenic species by applying the method to Mycobacterium bovis BCG cells, an attenuated strain of the M. tuberculosis complex.

Bovine serum albumin adsorption onto colloidal Al2O3 particles: a new model based on zeta potential and UV-vis measurements.

We investigated the adsorption of bovine serum albumin (BSA) on colloidal Al2O3 particles in an aqueous environment. Changes in the zeta potential of the Al2O3 particles upon the adsorption of BSA were measured using an electro-acoustic technique. The mass of protein adsorbed was determined by using UV-vis spectroscopy. The change of the isoelectric point of the Al2O3 powder-protein suspension was found to be a function of adsorbed protein mass. It was shown that approximately one monolayer of BSA was needed to fully mask the surface and to compromise the charge of Al2O3. From titration experiments it follows that about 30-36% of the negatively charged groups of the protein form bonds with the protonated and charged Al2O3 surface. On the basis of our observations we introduced a new adsorption model for BSA on Al2O3 particles.

A recA deletion mutant of Mycobacterium bovis BCG confers protection equivalent to that of wild-type BCG but shows increased genetic stability.

The widely used vaccine against tuberculosis, BCG, shows evidence of genetic instability. It has undergone major genetic rearrangements resulting in deletion and duplication of segments of its chromosome. In order to produce a BCG strain with more favourable genetic properties, we inactivated the recA gene. Targeted deletion of the recA gene of BCG resulted in a complete loss of recombination between homologous, chromosomally-located sequences, as well as between plasmid- and chromosomally-located sequences. The deltarecA mutant BCG was as effective as the wild-type in conferring protection in mice against an intravenous challenge with virulent Mycobacterium tuberculosis, indicating that the loss of an SOS response-mediated DNA repair mechanism did not compromise the immunological properties of BCG. The availability of a genetically stable, fully immunogenic BCG is important for the future development of BCG as a live vaccine.