TMKink: a method to predict transmembrane helix kinks

A hallmark of membrane protein structure is the large number of distorted transmembrane helices. Because of the prevalence of bends, it is important to not only understand how they are generated but also to learn how to predict their occurrence. Here, we find that there are local sequence preferences in kinked helices, most notably a higher abundance of proline, which can be exploited to identify bends from local sequence information. A neural network predictor identifies over two-thirds of all bends (sensitivity 0.70) with high reliability (specificity 0.89). It is likely that more structural data will allow for better helix distortion predictors with increased coverage in the future. The kink predictor, TMKink, is available at http://tmkinkpredictor.mbi.ucla.edu/.     

Novel terpenoids of the fungus Aspergillus insuetus isolated from the Mediterranean sponge Psammocinia sp. collected along the coast of Israel

Three novel meroterpenoids, insuetolides A-C (1-3) and four drimane sesquiterpenes, the new (E)-6-(4'-hydroxy-2'-butenoyl)-strobilactone A (4) and the known 2α, 9α, 11-trihydroxy-6-oxodrim-7-ene (5), strobilactone A (6) and (E,E)-6-(6',7'-dihydroxy-2',4'-octadienoyl)-strobilactone A (7), were isolated from the EtOAc extract of the culture medium of the marine-derived fungus Aspergillus insuetus (OY-207), which was isolated from the Mediterranean sponge Psammocinia sp. The structures of the compounds were determined by spectroscopic methods. Insuetolides A-C reveal a new carbon skeleton derived from the cyclization of farnesyl and 3, 5-dimethylorsellinic acid. Compounds 1, 6, and 7 exhibited anti-fungal activity towards Neurospora crassa with MIC values of 140, 242, and 162 μM, respectively; and compounds 3, 4, and 7 exhibited mild cytotoxicity towards MOLT-4 human leukemia cells.     

Modulating ALA-PDT efficacy of mutlidrug resistant MCF-7 breast cancer cells using ALA prodrug

Multi-drug resistance of breast cancer is a major obstacle in chemotherapy of cancer treatments. Recently it was suggested that photodynamic therapy (PDT) can overcome drug resistance of tumors. ALA-PDT is based on the administration of 5-aminolevulinic acid (ALA), the natural precursor for the PpIX biosynthesis, which is a potent natural photosensitizer. In the present study we used the AlaAcBu, a multifunctional ALA-prodrug for photodynamic inactivation of drug resistant MCF-7/DOX breast cancer cells. Supplementation of low doses (0.2mM) of AlaAcBu to the cells significantly increased accumulation of PpIX in both MCF-7/WT and MCF-7/DOX cells in comparison to ALA, or ALA + butyric acid (BA). In addition, our results show that MCF-7/DOX cells are capable of producing higher levels of porphyrins than MCF-7/WT cells due to low expression of the enzyme ferrochelatase, which inserts iron into the tetra-pyrrol ring to form the end product heme. Light irradiation of the AlaAcBu treated cells activated efficient photodynamic killing of MCF-7/DOX cells similar to the parent MCF-7/WT cells, depicted by low mitochondrial enzymatic activity, LDH leakage and decreased cell survival following PDT. These results indicate that the pro-drug AlaAcBu is an effective ALA derivative for PDT treatments of multidrug resistant tumors.     

Aerosols transmit prions to immunocompetent and immunodeficient mice

Prions, the agents causing transmissible spongiform encephalopathies, colonize the brain of hosts after oral, parenteral, intralingual, or even transdermal uptake. However, prions are not generally considered to be airborne. Here we report that inbred and crossbred wild-type mice, as well as tga20 transgenic mice overexpressing PrP(C), efficiently develop scrapie upon exposure to aerosolized prions. NSE-PrP transgenic mice, which express PrP(C) selectively in neurons, were also susceptible to airborne prions. Aerogenic infection occurred also in mice lacking B- and T-lymphocytes, NK-cells, follicular dendritic cells or complement components. Brains of diseased mice contained PrP(Sc) and transmitted scrapie when inoculated into further mice. We conclude that aerogenic exposure to prions is very efficacious and can lead to direct invasion of neural pathways without an obligatory replicative phase in lymphoid organs. This previously unappreciated risk for airborne prion transmission may warrant re-thinking on prion biosafety guidelines in research and diagnostic laboratories.     

Pre-clinical and clinical significance of heparanase in Ewing's sarcoma

Heparanase is an endoglycosidase that specifically cleaves heparan sulphate side chains of heparan sulphate proteoglycans, activity that is strongly implicated in cell migration and invasion associated with tumour metastasis, angiogenesis and inflammation. Heparanase up-regulation was documented in an increasing number of human carcinomas, correlating with reduced post-operative survival rate and enhanced tumour angiogenesis. Expression and significance of heparanase in human sarcomas has not been so far reported. Here, we applied the Ewing's sarcoma cell line TC71 and demonstrated a potent inhibition of cell invasion in vitro and tumour xenograft growth in vivo upon treatment with a specific inhibitor of heparanase enzymatic activity (compound SST0001, non-anticoagulant N-acetylated, glycol split heparin). Next, we examined heparanase expression and cellular localization by immunostaining of a cohort of 69 patients diagnosed with Ewing's sarcoma. Heparanase staining was noted in all patients. Notably, heparanase staining intensity correlated with increased tumour size (P = 0.04) and with patients' age (P = 0.03), two prognostic factors associated with a worse outcome. Our study indicates that heparanase expression is induced in Ewing's sarcoma and associates with poor prognosis. Moreover, it encourages the inclusion of heparanase inhibitors (i.e. SST0001) in newly developed therapeutic modalities directed against Ewing's sarcoma and likely other malignancies.     

Unchanged glutamine synthetase activity and increased NMDA receptor density in epileptic human neocortex: implications for the pathophysiology of epilepsy

We investigated whether alterations in glutamate metabolising glutamine synthetase activity occur in human epileptic neocortex, as shown previously for human epileptic hippocampus [Eid, T., Thomas, M.J., Spencer, D.D., Rundén-Pran, E., Lai, J.C.K., Malthankar, G.V., Kim, J.H., Danbolt, N.C., Ottersen, O.P., de Lanerolle, N.C., 2004. Loss of glutamine synthetase in the human epileptic hippocampus: possible mechanism for raised extracellular glutamate in mesial temporal lobe epilepsy. Lancet 363, 28-37]. Glutamine synthetase activity was equivalent in both non-epileptic and epileptic human neocortex. Epileptic tissue, however, was characterised by a 37% increase in the density of synaptosomal NMDA receptor sites compared to non-epileptic tissue, as revealed by a radioligand binding assay (B max(non-epileptic) 1.45 pmol/mg protein and B max(epileptic) 1.99 pmol/mg protein, P < 0.05). Our findings shed some doubts on a role of glutamine synthetase in the pathophysiology of epilepsy in the neocortex. However, the detection of a significantly reduced enzymatic activity in the epileptic amygdala supports the assumption that the enzyme defect is localized to the epileptic mesial temporal lobe of corresponding patients.     

Phosphoproteomic analysis using immobilized metal ion affinity chromatography on the basis of cellulose powder

Detailed characterization of phosphoproteins as well as other post-translationally modified proteins such as glycoproteins, is required to fully understand protein function and regulatory events in cells and organisms. Therefore, an experimental strategy for the isolation of phosphoproteins using a new immobilized metal ion affinity chromatograph (IMAC) material on the basis of cellulose has been developed and characterized. Different approaches have been used to test the material. Recovery rates were determined by 32P labelling of a myelin basic protein fragment and by reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry using a tryptic digest of the model protein bovine beta-casein. Selectivity was demonstrated by enrichment and separation of phosphopeptides from different samples, such as from a digest of horse myoglobin as well as from a digest of in vitro phosphorylated extracellular signal regulates kinase 2 (ERK2) mixed with synthetic phosphopeptides, phosphorylated on different amino acid residues. Furthermore, simplification and optimization of sample pretreatment was achieved by combining the separating (IMAC) and desalting (C18) step during preparative high performance liquid chromatography. The comparison between our material and a commercially available IMAC system (POROS 20 MC; Perspective BioSystems) emphasizes the competitiveness of the cellulose. Confirmed by the obtained data, the cellulose material performed as well as the commercially available sorbent, however with the advantage, that it can be produced rather easily and at very low cost.     

Evaluation of prefractionation methods as a preparatory step for multidimensional based chromatography of serum proteins

Prefractionations of proteins prior to their proteolysis, chromatography, and MS/MS analyses help reduce complexity and increase the yield of protein identifications. A number of methods were evaluated here for prefractionating serum samples distributed to the participating laboratories as part of the human Plasma Proteome Project. These methods include strong cation exchange (SCX) chromatography, slicing of SDS-PAGE gel bands, and liquid-phase IEF of the proteins. The fractionated proteins were trypsinized and the resulting peptides were resolved and analyzed by multidimensional protein identification technology coupled to IT MS/MS. The MS/MS spectra were clustered, combined, and searched against the IPI protein databank using Pep-Miner. The identification results were evaluated for the efficacy of the different prefractionation methodologies to identify larger numbers of proteins at higher confidence and to achieve the best coverage of the proteins with the identified peptides. Prefractionation based on SCX resulted in the largest number of identified proteins, followed by gel slices and then the liquid-phase IEF. An important observation was that each of the methods revealed a set of unique proteins, some identified with high confidence. Therefore, for comprehensive identification of the serum proteins, several different prefractionation approaches should be used in parallel.     

Common features in the functional surface of scorpion beta-toxins and elements that confer specificity for insect and mammalian voltage-gated sodium channels

Scorpion beta-toxins that affect the activation of mammalian voltage-gated sodium channels (Navs) have been studied extensively, but little is known about their functional surface and mode of interaction with the channel receptor. To enable a molecular approach to this question, we have established a successful expression system for the anti-mammalian scorpion beta-toxin, Css4, whose effects on rat brain Navs have been well characterized. A recombinant toxin, His-Css4, was obtained when fused to a His tag and a thrombin cleavage site and had similar binding affinity for and effect on Na currents of rat brain sodium channels as those of the native toxin isolated from the scorpion venom. Molecular dissection of His-Css4 elucidated a functional surface of 1245 A2 composed of the following: 1) a cluster of residues associated with the alpha-helix, which includes a putative "hot spot" (this cluster is conserved among scorpion beta-toxins and contains their "pharmacophore"); 2) a hydrophobic cluster associated mainly with the beta2 and beta3 strands, which is likely to confer the specificity for mammalian Navs; 3) a single bioactive residue (Trp-58) in the C-tail; and 4) a negatively charged residue (Glu-15) involved in voltage sensor trapping as inferred from our ability to uncouple toxin binding from activity upon its substitution. This study expands our understanding about the mode of action of scorpion beta-toxins and illuminates differences in the functional surfaces that may dictate their specificities for mammalian versus insect sodium channels.