Establishment of a two-dimensional electrophoresis map for Neospora caninum tachyzoites by proteomics

Expressed proteins and antigens from Neospora caninum tachyzoites were studied by two-dimensional gel electrophoresis and immunoblot analysis combined with matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Thirty-one spots corresponding to 20 different proteins were identified from N. caninum tachyzoites by peptide mass fingerprinting. Six proteins were identified from a N. caninum database (NTPase, 14-3-3 protein homologue, NcMIC1, NCDG1, NcGRA1 and NcGRA2), and 11 proteins were identified in closely related species using the T. gondii database (HSP70, HSP60, pyruvate kinase, tubulin alpha- and beta-chain, putative protein disulfide isomerase, enolase, actin, fructose-1,6-bisphosphatase, lactate dehydrogenase and glyceradehyde-3-phosphate dehydrogenase). One hundred and two antigen spots were observed using pH 4-7 IPG strips on immunoblot profiles. Among them, 17 spots corresponding to 11 antigenic proteins were identified from a N. caninum protein map. This study involved the construction of in-depth protein maps for N. caninum tachyzoites, which will be of value for studies of its pathogenesis, drug and vaccine development, and phylogenetic studies.     

Sphingosine and FTY720 modulate pacemaking activity in interstitial cells of Cajal from mouse small intestine

Interstitial cells of Cajal (ICCs) are the pacemakers of the gastrointestinal tract, and transient receptor potential melastatin type 7 (TRPM7) and Ca²⁺ activated Cl⁻ channels (ANO1) are candidate the generators of pacemaker potentials in ICCs. The effects of D-erythro-sphingosine (SPH) and structural analogues of SPH, that is, N,N-dimethyl-Derythro-sphingosine (N,N-DMS), FTY720, and FTY720-P on the pacemaking activities of ICCs were examined using the whole cell patch clamp technique. SPH, N,N-DMS, and FTY720 decreased the amplitudes of pacemaker potentials in ICC clusters, but resting membrane potentials displayed little change. Also, perfusing SPH, N,N-DMS, or FTY720 in the bath reduced both inward and outward TRPM7-like currents in single ICCs, and inhibited ANO1 currents. The another structural analogue of SPH, FTY720-P was ineffective at the pacemaker potentials in ICC clusters and the TRPM7-like currents in single ICCs. Furthermore, FTY720- P had no effect on ANO1. These results suggest that SPH, N,N-DMS, and FTY720 modulate the pacemaker activities of ICCs, and that TRPM7 and ANO1 channels affect intestinal motility.     

Targeted delivery of a phosphopeptide prodrug inhibits the proliferation of a human glioma cell line

Peptides are ideal candidates for developing therapeutics. Polo-like kinase 1 is an important regulatory protein in the cell cycle and contains a C-terminal polo-box domain, which is the hallmark of this protein family. We developed a peptide inhibitor of polo-like kinase 1 that targets its polo-box domain. This new phosphopeptide, cRGDyK-S-S-CPLHSpT, preferentially penetrates the cancer cell membrane mediated by the integrin receptor, which is expressed at high levels by cancer cells. In the present study, using high performance liquid chromatography and mass spectroscopy, we determined the stability of cRGDyK-S-S-CPLHSpT and its cleavage by glutathione under typical conditions for cell culture. We further assessed the ability of the peptide to inhibit the proliferation of the U87MG glioma cell line. The phosphorylated peptide was stable, and the disulfide bond of cRGDyK-S-S-CPLHSpT was cleaved in 50 mM glutathione. This peptide inhibited the growth of cancer cells and changed their morphology. Therefore, we conclude that the phosphopeptide shows promise as a prodrug and has a high potential to act as an anticancer agent by inhibiting polo-like kinase 1 by binding its polo-box domain. These findings indicate the therapeutic potential of PLHSpT and peptides similarly targeted to surface receptors of cancer cells and to the functional domains of regulatory proteins.     

Down regulation of extramacrochaetae mRNA by a Drosophila neural RNA binding protein Rbp9 which is homologous to human Hu proteins.

Rbp9 is an RNA binding protein expressed mainly in the central nervous system of adult Drosophilamelanogaster. Rbp9 shares a high degree of sequence similarity with human neural proteins referred to as Hu antigens. Hu antigens bind to U-rich mRNA destabilizing elements with a high affinity and, thus, have been implicated as regulators of mRNA stability. Using in vitro RNA binding assays, we found that Rbp9 binds strongly to poly U sequences. We then employed a Selex system to identify a consensus Rbp9 binding site (UUUXUUUU). Information obtained from the Selex results allowed the detection of two repeats of the Rbp9 consensus binding sequence in the 3' untranslated region of extramacrochaetae mRNA. UV crosslinking experiments demonstrated that Rbp9 interactsspecifically with emc mRNA. The requirement of Rbp9 protein in the down regulation of emc mRNA was confirmed by northern (RNA) analysis, which revealed that the level of emc mRNA increased 10-fold in rbp9 mutant flies. Taken together with the in vitro RNA binding results, the genetic evidence obtained strongly supports the hypothesis that Rbp9 functions as a regulator of RNA stability.     

Identification of a novel system L amino acid transporter structurally distinct from heterodimeric amino acid transporters

A cDNA that encodes a novel Na+-independent neutral amino acid transporter was isolated from FLC4 human hepatocarcinoma cells by expression cloning. When expressed in Xenopus oocytes, the encoded protein designated LAT3 (L-type amino acid transporter 3) transported neutral amino acids such as l-leucine, l-isoleucine, l-valine, and l-phenylalanine. The LAT3-mediated transport was Na+-independent and inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid, consistent with the properties of system L. Distinct from already known system L transporters LAT1 and LAT2, which form heterodimeric complex with 4F2 heavy chain, LAT3 was functional by itself in Xenopus oocytes. The deduced amino acid sequence of LAT3 was identical to the gene product of POV1 reported as a prostate cancer-up-regulated gene whose function was not determined, whereas it did not exhibit significant similarity to already identified transporters. The Eadie-Hofstee plots of LAT3-mediated transport were curvilinear, whereas the low affinity component is predominant at physiological plasma amino acid concentration. In addition to amino acid substrates, LAT3 recognized amino acid alcohols. The transport of l-leucine was electroneutral and mediated by a facilitated diffusion. In contrast, l-leucinol, l-valinol, and l-phenylalaninol, which have a net positive charge induced inward currents under voltage clamp, suggesting these compounds are transported by LAT3. LAT3-mediated transport was inhibited by the pretreatment with N-ethylmaleimide, consistent with the property of system L2 originally characterized in hepatocyte primary culture. Based on the substrate selectivity, affinity, and N-ethylmaleimide sensitivity, LAT3 is proposed to be a transporter subserving system L2. LAT3 should denote a new family of organic solute transporters.     

Characterization of ELP-fused ω-Transaminase and Its Application for the Biosynthesis of β-Amino Acid

Optically pure amines, β-amino acids and γ-amino acids are the valuable precursors to produce biologically active compounds. The ω-TAs are the class of enzymes which are widely used to produce such compounds. In this work (S)-ω-transaminase from the thermophilic eubacterium Sphaerobacter thermophilus (St-TA) was fused with Elastin-like polypeptides (ELPs) through the cloning process and expressed in E. coli cells. The characterization of this fusion complex was performed with respect to thermostability and effect of DMSO. Where in case of St-TA-ELP-V₆₀, major difference in the transition temperature (T_t) was observed, wherein a T_t of 38 and 70°C was observed at the increasing concentration of DMSO from 5 to 25% (v/v). Interestingly, these fusion proteins the activity was preserved even after the aggregation of fusion complex at Tt. The substrate specificity and product inhibition analysis showed that ω-TA-ELPs had comparable results as that of wild type ω-TA. Moreover, the fused ω-TA could be efficiently reused for up to 20 batches of transamination reaction. Furthermore, the applicability of the fusion protein for the production of a sitagliptin precursor (R)-3-amino-4-(2,4,5-triflurophenyl) butanoic acid (3-ATfBA) was evaluated, wherein 3-ATfBA was synthesized with good conversion (65%).