Identification of TH1 as an interaction partner of A-Raf kinase

A-Raf is an important intermediate of the growth factor Ras-MAP kinase pathway. In a two-hybrid screen of human fetal liver cDNA library, TH1 was detected as a new interaction partner of A-Raf. TH1 is a highly conserved and widely expressed protein, which was recently cloned by Bonthron DT group. The binding between A-Raf and TH1 was specific, as no binding between TH1 and B-Raf or c-Raf was observed, and the amino-terminal 162 amino acids in the A-Raf regulatory domain were found to be sufficient for this interaction. This specific interaction may have played a critical role in the activation of A-Raf.     

Route of administration of chimeric BPV1 VLP determines the character of the induced immune responses

To examine the mucosal immune response to papillomavirus virus-like particles (PV-VLP), mice were immunized with VLP intrarectally (i.r.), intravaginally (i.va.) or intramuscularly (i.m.) without adjuvant. PV-VLP were assembled with chimeric BPV-1 L1 proteins incorporating sequence from HIV-1 gp120, either the V3 loop or a shorter peptide incorporating a known CTL epitope (HIVP18I10). Antibody specific for BPV-1 VLP and P18 peptide was detected in serum following i.m., but not i.r. or i.va. immunization. Denatured VLP induced a much reduced immune response when compared with native VLP. Immune responses following mucosal administration of VLP were generally weaker than following systemic administration. VLP specific IgA was higher in intestine washes following i.r. than i.va. immunization, and higher in vaginal washes following i.m. than i.r. or i.va. immunization. No differences in specific antibody responses were seen between animals immunized with BPV-1 P18 VLP or with BPV-1 V3 VLP. Cytotoxic T lymphocyte precursors specific for the P18 CTL epitope were recovered from the spleen following i.m., i.va. or i.r. immunization with P18 VLP, and were similarly detected in Peyer's patches following i.m. or i.r. immunization. Thus, mucosal or systemic immunization with PV VLP induces mucosal CTL responses and this may be important for vaccines for mucosal infection with human papillomaviruses and for other viruses.     

Correlation between hydrophobic properties and efficiency of carrier-mediated membrane transduction and apoptosis of a p53 C-terminal peptide

Two membrane transporters, the 17 amino acid (aa) oligopeptide penetratin derived from the homeodomain of Antennapedia (Ant) and an analogue of the basic domain of TAT (aa 47-57) (TAT-a) from HIV-1, were tested as carriers for a p53 C-terminal peptide (aa 361-382) into human breast cancer cells. The studies were performed to determine whether the membrane-transduction efficiency of membrane carriers: Ant, TAT or TAT analogue (TAT-a) correlated with peptide hydrophobic features. Peptide-sequence analysis clearly demonstrated that the Ant sequence and p53 peptide sequence (p53p) together created a peptide with enhanced hydrophobic characteristics; while the TAT or TAT analogue (TAT-a) and p53p sequence together created a peptide with significantly less hydrophobic qualities. The degree of hydrophobic moment and helical wheel plots for these peptides correlated directly with their ability to transduce the p53 peptide. Western blot analysis revealed that Ant was able to transduce p53 C-terminal peptide into human breast cancer cells as a highly efficient membrane transporter. Compared to Ant, TAT-a fused to the C-terminus of p53 peptide (p53p-TAT-a) was a less efficient carrier into these cells under the conditions of our study. Additionally, N-terminal linked TAT-a to p53p (TAT-a-p53p) showed even lower efficiency as a transporter than p53-TAT-a. Apoptosis assays showed that the p53 peptide, fused at its C-terminus to Ant (p53p-Ant), induced a higher percentage of apoptotic cells in human breast cancer cell lines expressing mutant or wild-type p53 as compared to p53 peptide fused at its C-terminus to the TAT-a sequence (p53p-TAT-a) or when fused at the N-terminus to TAT-a (TAT-a-p53p). These data suggested a direct correlation between hydrophobic characteristics and efficiency as a transporter. Sequence study, using hydrophobic moment and helical wheel analyses, may be useful predictive tools for choosing the best carrier for a peptide.     

Human serum and glucocorticoid-inducible kinase-like kinase (SGKL) phosphorylates glycogen syntheses kinase 3 beta (GSK-3beta) at serine-9 through direct interaction

Serum and glucocorticoid-inducible kinase-like kinase (SGKL) has been identified as a new integrator that decodes lipid signals produced by the activation of phosphoinositide 3-kinase (PI3K). SGKL is activated via its lipid-binding domain (phox homology domain) in response to PI3K signaling. However, downstream targets of SGKL as well as the role of SGKL as a mediator in PI3K signaling in human tissues remain to be established. In this study, we identified human glycogen synthase kinase 3 beta (GSK-3beta) as a specific interacting partner with SGKL in a yeast two-hybrid screening of human brain cDNA library. The association between these two proteins is confirmed independently in human embryonic kidney (HEK293) cells by co-immunoprecipitation. Furthermore, the kinase activity of wild-type SGKL was required for the in vitro phosphorylation of a GSK-3 crosstide fusion protein at serine-21/9 as demonstrated with a Phospho-GSK-3alpha/beta (Ser21/9) specific antibody. The present results provide strong evidences that SGKL could utilize GSK-3beta as a direct downstream target by phosphorylating GSK-3beta at serine-9.     

Fe(2+)-catalyzed oxidation and cleavage of sarcoplasmic reticulum ATPase reveals Mg(2+) and Mg(2+)-ATP sites

Fe(2+) can substitute for Mg(2+) in activation of the sarcoplasmic reticulum (SR) ATPase, permitting approximately 25% activity in the presence of Ca(2+). Therefore, we used Fe(2+) to obtain information on the binding sites for Mg(2+) and the Mg(2+)-ATP complex within the enzyme structure. When the ATPase is incubated with Fe(2+) in the presence of H(2)O(2) and/or ascorbate, specific patterns of Fe(2+)-catalyzed oxidation and cleavage are observed in the SR ATPase, depending on its Ca(2+)-bound (E1-Ca(2)) or Ca(2+)-free conformation (E2-TG), as well as on the presence of ATP. The ATPase protein in the E1-Ca(2) state is cleaved efficiently by Fe(2+) with H(2)O(2) and ascorbate assistance, yielding a 70-75 kDa carboxyl end fragment. Cleavage of the ATPase protein in the E2-TG state occurs within the same region, but with a more diffuse pattern, yielding multiple fragments within the 65-85 kDa range. When Fe(2+) catalysis is assisted by ascorbate only (in the absence of H(2)O(2)), cleavage at the same protein site occurs much more slowly, and is facilitated by ATP (or AMP-PNP) and Ca(2+). Amino acid sequencing indicates that protein cleavage occurs at and near Ser346, and is attributed to Fe(2+) bound to a primary Mg(2+) site near Ser346 and neighboring Glu696. In addition, incubation with Fe(2+) and ascorbate produces Ca(2+)- and ATP-dependent oxidation of the Thr441 side chain, as demonstrated by NaB(3)H(4) incorporation and analysis of fragments obtained by extensive trypsin digestion. This oxidation is attributed to bound Fe(2+)-ATP complex, as shown by structural modeling of the Mg(2+)-ATP complex at the substrate site.     

Type II phosphatidylinositol 4-kinase beta is a cytosolic and peripheral membrane protein that is recruited to the plasma membrane and activated by Rac-GTP

Phosphoinositides have a pivotal role as precursors to important second messengers and as bona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIalpha and PI4KIIbeta. PI4KIIalpha was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIbeta and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIbeta is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIalpha is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIbeta recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIbeta translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIalpha further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIbeta activation, because microsome-bound PI4KIIbeta is 16 times more active than cytosolic PI4KIIbeta. Membrane-associated PI4KIIbeta is as active as membrane-associated PI4KIIalpha and has essentially identical kinetic properties. We conclude that PI4KIIalpha and PI4KIIbeta may have partially overlapping, but not identical, functions. PI4KIIbeta is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells.     

Expression of ssDNA in mammalian cells

Antisense therapy involves the use of antisense oligonucleotides for altering targeted gene function. However, the low efficiency of cell delivery of antisense oligonucleotides has limited the efficacy of antisense therapeutic approaches. RNA-based antisense or ribozyme oligonucleotides can be either synthesized endogenously (e.g., by a viral vector) or delivered exogenously. However, there is presently no vector delivery system available for DNA-based oligonucleotides. Recently, a novel ssDNA expression vector that can generate intracellularly any ssDNA molecule, such as antisense oligonucleotide or DNA enzyme, has been developed in our laboratory. Here we describe an improved expression vector based on the first-generation two-vector system. To test this new expression vector, we chose to express a single-stranded "10-23" DNA enzyme targeting c-raf mRNA in the human lung carcinoma A549 cell line. After introduction into cells by transient transfection, c-raf-cleaving DNA enzymes produced by this expression vector can significantly suppress the expression of c-raf mRNA. Furthermore, the expressed c-raf DNA enzymes induced cell apoptosis, as indicated by genomic DNA fragmentation assay. Our study further demonstrates the feasibility of using this novel ssDNA expression technology to produce intracellularly any sequence of interest, including antisense oligonucleotides and DNA enzyme molecules.     

Phosphatidylinositol 4 phosphate regulates targeting of clathrin adaptor AP-1 complexes to the Golgi

Phosphatidylinositol 4 phosphate [PI(4)P] is essential for secretion in yeast, but its role in mammalian cells is unclear. Current paradigms propose that PI(4)P acts primarily as a precursor to phosphatidylinositol 4,5 bisphosphate (PIP2), an important plasma membrane regulator. We found that PI(4)P is enriched in the mammalian Golgi, and used RNA interference (RNAi) of PI4KIIalpha, a Golgi resident phosphatidylinositol 4 kinase, to determine whether PI(4)P directly regulates the Golgi. PI4KIIalpha RNAi decreases Golgi PI(4)P, blocks the recruitment of clathrin adaptor AP-1 complexes to the Golgi, and inhibits AP-1-dependent functions. This AP-1 binding defect is rescued by adding back PI(4)P. In addition, purified AP-1 binds PI(4)P, and anti-PI(4)P inhibits the in vitro recruitment of cytosolic AP-1 to normal cellular membranes. We propose that PI4KIIalpha establishes the Golgi's unique lipid-defined organelle identity by generating PI(4)P-rich domains that specify the docking of the AP-1 coat machinery.     

Application of a two-stage temperature control strategy for enhanced glutathione production in the batch fermentation by Candida utilis

In batch culture for glutathione production with Candida utilis, a higher temperature (30 °C) was required to hasten cell growth while a lower temperature (26 °C) was needed to increase the production of glutathione. A two-stage temperature control strategy was used to enhance both the yield and the productivity of glutathione. As a result, glutathione production was increased by 5% and 23% of that at 26 °C and 30 °C, respectively, and the intracellular glutathione content reached 2.5% (w/w).