Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

Peptide toxins found in a wide array of venoms block K⁺ channels, causing profound physiological and pathological effects. Here we describe the first functional K⁺ channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23_TxD) that is evolutionarily related to peptide toxins from sea anemones. MMP23_TxD shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K⁺ channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K⁺ channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K⁺ channels by co-localizing with and trapping MMP23_TxD-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.     

Cosecretion of protease inhibitor stabilizes antibodies produced by plant roots

A plant-based system for continuous production of monoclonal antibodies based on the secretion of immunoglobulin complexes from plant roots into a hydroponic medium (rhizosecretion) was engineered to produce high levels of single-chain and full-size immunoglobulins. Replacing the original signal peptides of monoclonal antibodies with a plant-derived calreticulin signal increased the levels of antibody yield 2-fold. Cosecretion of Bowman-Birk Ser protease inhibitor reduced degradation of the immunoglobulin complexes in the default secretion pathway and further increased antibody production to 36.4 microg/g root dry weight per day for single-chain IgG1 and 21.8 microg/g root dry weight per day for full-size IgG4 antibodies. These results suggest that constitutive cosecretion of a protease inhibitor combined with the use of the plant signal peptide and the antibiotic marker-free transformation system offers a novel strategy to achieve high yields of complex therapeutic proteins secreted from plant roots.     

Presenilins form ER Ca2+ leak channels, a function disrupted by familial Alzheimer's disease-linked mutations

Alzheimer's disease (AD) is a progressive and irreversible neurodegenerative disorder. Mutations in presenilins 1 and 2 (PS1 and PS2) account for approximately 40% of familial AD (FAD) cases. FAD mutations and genetic deletions of presenilins have been associated with calcium (Ca(2+)) signaling abnormalities. We demonstrate that wild-type presenilins, but not PS1-M146V and PS2-N141I FAD mutants, can form low-conductance divalent-cation-permeable ion channels in planar lipid bilayers. In experiments with PS1/2 double knockout (DKO) mouse embryonic fibroblasts (MEFs), we find that presenilins account for approximately 80% of passive Ca(2+) leak from the endoplasmic reticulum. Deficient Ca(2+) signaling in DKO MEFs can be rescued by expression of wild-type PS1 or PS2 but not by expression of PS1-M146V or PS2-N141I mutants. The ER Ca(2+) leak function of presenilins is independent of their gamma-secretase activity. Our data suggest a Ca(2+) signaling function for presenilins and provide support for the "Ca(2+) hypothesis of AD."     

Detection and quantification of Legionella pneumophila in water samples using competitive PCR

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.     

Rotamer libraries and probabilities of transition between rotamers for the side chains in protein-protein binding

Conformational changes in the side chains are essential for protein-protein binding. Rotameric states and unbound- to-bound conformational changes in the surface residues were systematically studied on a representative set of protein complexes. The side-chain conformations were mapped onto dihedral angles space. The variable threshold algorithm was developed to cluster the dihedral angle distributions and to derive rotamers, defined as the most probable conformation in a cluster. Six rotamer libraries were generated: full surface, surface noninterface, and surface interface-each for bound and unbound states. The libraries were used to calculate the probabilities of the rotamer transitions upon binding. The stability of amino acids was quantified based on the transition maps. The noninterface residues' stability was higher than that of the interface. Long side chains with three or four dihedral angles were less stable than the shorter ones. The transitions between the rotamers at the interface occurred more frequently than on the noninterface surface. Most side chains changed conformation within the same rotamer or moved to an adjacent rotamer. The highest percentage of the transitions was observed primarily between the two most occupied rotamers. The probability of the transition between rotamers increased with the decrease of the rotamer stability. The analysis revealed characteristics of the surface side-chain conformational transitions that can be utilized in flexible docking protocols.     

Identification of candidate SNPs for drug induced toxicity from differentially expressed genes in associated tissues

The growing collection of publicly available high-throughput data provides an invaluable resource for generating preliminary in silico data in support of novel hypotheses. In this study we used a cross-dataset meta-analysis strategy to identify novel candidate genes and genetic variations relevant to paclitaxel/carboplatin-induced myelosuppression and neuropathy. We identified genes affected by drug exposure and present in tissues associated with toxicity. From ten top-ranked genes 42 non-synonymous single nucleotide polymorphisms (SNPs) were identified in silico and genotyped in 94 cancer patients treated with carboplatin/paclitaxel. We observed variations in 11 SNPs, of which seven were present in a sufficient frequency for statistical evaluation. Of these seven SNPs, three were present in ABCA1 and ATM, and showed significant or borderline significant association with either myelosuppression or neuropathy. The strikingly high number of associations between genotype and clinically observed toxicity provides support for our data-driven computations strategy to identify biomarkers for drug toxicity.