The Tat system proofreads FeS protein substrates and directly initiates the disposal of rejected molecules

The twin-arginine translocation (Tat) system transports folded proteins across the bacterial plasma membrane, including FeS proteins that receive their cofactors in the cytoplasm. We have studied two Escherichia coli Tat substrates, NrfC and NapG, to examine how, or whether, the system exports only correctly folded and assembled FeS proteins. With NrfC, substitutions in even one of four predicted FeS centres completely block export, indicating an effective proofreading activity. The FeS mutants are rapidly degraded but only if they interact with the Tat translocon; they are stable in a tat deletion strain and equally stable in wild-type cells if the signal peptide twin-arginine motif is removed to block targeting. Basically similar results are obtained with NapG. The Tat apparatus thus proofreads these substrates and directly initiates the turnover of rejected molecules. Turnover of mutated FeS substrates is completely dependent on the TatA/E subunits that are believed to be involved in the late stages of translocation, and we propose that partial translocation triggers substrate turnover within an integrated quality control system for FeS proteins.     

A p38 MAPK-CREB pathway functions to pattern mesoderm in Xenopus

Dorsal-ventral patterning is specified by signaling centers secreting antagonizing morphogens that form a signaling gradient. Yet, how morphogen gradient is translated intracellularly into fate decisions remains largely unknown. Here, we report that p38 MAPK and CREB function along the dorsal-ventral axis in mesoderm patterning. We find that the phosphorylated form of CREB (S133) is distributed in a gradient along the dorsal-ventral mesoderm axis and that the p38 MAPK pathway mediates the phosphorylation of CREB. Knockdown of CREB prevents chordin expression and mesoderm dorsalization by the Spemann organizer, whereas ectopic expression of activated CREB-VP16 chimera induces chordin expression and dorsalizes mesoderm. Expression of high levels of p38 activator, MKK6E or CREB-VP16 in embryos converts ventral mesoderm into a dorsal organizing center. p38 MAPK and CREB function downstream of maternal Wnt/β-catenin and the organizer-specific genes siamois and goosecoid. At low expression levels, MKK6E induces expression of lateral genes without inducing the expression of dorsal genes. Loss of CREB or p38 MAPK activity enables the expansion of the ventral homeobox gene vent1 into the dorsal marginal region, preventing the lateral expression of Xmyf5. Overall, these data indicate that dorsal-ventral mesoderm patterning is regulated by differential p38/CREB activities along the axis.     

Thermodynamic contributions of the reactions of DNA intramolecular structures with their complementary strands

One focus of our research is to further our understanding of the physico-chemical properties of unusual DNA structures and their interaction with complementary oligonucleotides. We have investigated three types of reactions involving the interaction of intramolecular DNA complexes with their complementary single strands of varied length. Specifically, we have used a combination of isothermal titration (ITC) and differential scanning (DSC) calorimetry and spectroscopy techniques to determine standard thermodynamic profiles for the reaction of an i-motif, G-quadruplex, and triplex with their complementary strands. The enthalpies for each reaction are measured directly in ITC titrations and compared with those obtained indirectly from Hess cycles using DSC unfolding data. All reactions investigated yielded favorable free energy contributions, indicating that each single strand is able to invade and disrupt the corresponding intramolecular DNA complex. These favorable free energy terms are enthalpy driven, which result from a compensation of exothermic contributions, due to the formation of additional base-pair stacks (or base-triplet stacks) in the duplex product (or triplex product), immobilization of electrostricted water by the base-pair and base-triplet stacks, and the removal of structural water from the reactant single strands; and endothermic contributions from the disruption of base-base stacking interactions of the reactant single strands. This investigation of nucleic acid reactions has provided new methodology, based on physico-chemical principles, to determine the molecular forces involved in the interactions between DNA nucleic acid structures. This methodology may be used in targeting reactions for the control of gene expression.     

Enhancement of gene transfer using YIGSR analog of Tat-derived peptide

Cell penetrating peptide based gene carriers are notably known for low level of gene transfer. To remedy this, as laminin receptor (LR) has been previously linked to tumor metastasis, the LR-binding domain (YIGSR) as well as a scrambled sequence (SGIYR) were added to Tat-derived peptide sequence (YIGSR-Tat and SGIYR-Tat respectively). Peptides cellular uptake was assessed with high-LR (HT1080) and low-LR (HT29) cell lines by flow cytometry. Their ability to form complexes with DNA was examined using YOPRO-1 fluorescence assay and their transfection efficiencies evaluated using a luciferase reporter gene assay. DNA complexes were formed at (+/-) charge ratios as low as 2:1. While no conclusion could be drawn on the effect of YIGSR sequence on peptides uptake in both cell lines, a significant improvement in gene transfection in HT1080 cells was achieved using YIGSR-Tat compared to Tat and SGIYR-Tat. Additionally this increased efficiency was inhibited by excess free YIGSR. No significant difference in transfection efficiency was observed between Tat, SGIYR-Tat and YIGSR-Tat based complexes in HT29 cells. These studies demonstrate that attachment of receptor-binding ligand (YIGSR) to Tat-derived peptide can improve the efficiency of gene transfer in LR-positive cells (HT1080).     

Prostate Apoptosis Response-4 Mediates Trophic Factor Withdrawal-Induced Apoptosis of Hippocampal Neurons

Prostate apoptosis response-4 (Par-4) is the product of a gene up-regulated in prostate cancer cells undergoing apoptosis. We now report that Par-4 mRNA and protein levels rapidly and progressively increase 4-24 h following trophic factor withdrawal (TFW) in cultured embryonic rat hippocampal neurons. The increased Par-4 levels follow an increase of reactive oxygen species, and precede mitochondrial membrane depolarization, caspase activation, and nuclear chromatin condensation/fragmentation. Pretreatment of cultures with 17beta-estradiol, vitamin E, and uric acid largely prevented Par-4 induction and cell death following TFW, demonstrating necessary roles for oxidative stress and membrane lipid peroxidation in TFW-induced neuronal apoptosis. Par-4 antisense oligonucleotide treatment blocked Par-4 protein increases and attenuated mitochondrial dysfunction, caspase activation, and cell death following TFW. Collectively, our data identify Par-4 as an early and pivotal player in neuronal apoptosis resulting from TFW and suggest that estrogen and antioxidants may prevent apoptosis, in part, by suppressing Par-4 production.     

Interferon γ and antisense transforming growth factor β transgenes synergize to enhance the immunogenicity of a murine mammary carcinoma

Transforming growth factor β (TGFβ) is an immunosuppressive cytokine that contributes to the immunological escape of tumor cells. In a previous study we demonstrated that inhibition of TGFβ production by EMT6 murine mammary tumor cells expressing an antisense TGF-β transgene reduces their tumorigenicity. On the basis of this observation we hypothesized that down-regulation of TGFβ production coupled with interferon γ (IFNγ) stimulation would induce an immune response superior to that generated by either strategy alone. In this study, EMT6 tumor cells expressing antisense TGFβ were transduced with the murine IFNγ gene. Tumor cells expressing either or both transgenes grew more slowly than mock-transduced tumors. Dual-transgene-expressing tumor cells were more immunogenic than tumor cells expressing either transgene alone. Studies in mice depleted of T cell subsets indicated that CD8⁺ T cells are the primary effectors of the antitumor activity observed. These results suggest that down-regulation of immunosuppression combined with cytokine-mediated immune augmentation is a useful strategy to improve antitumor immunity. Received: 6 October 1998 / Accepted: 15 January 1999     

The Influence of the Non‐Nucleotide Insert on the Hybridization Properties of Oligonucleotides

The effect of different non‐nucleotide inserts incorporated into oligonucleotide chains on their hybridization properties was studied by the method of thermal denaturation. Various types of alkyldiols and oligoethylene glycols were used as inserts modifying oligonucleotide backbone. Such modification of oligonucleotides caused the destabilization of their complementary complexes. It was shown that the hybridization properties of the modified oligonucleotides depend on several features of inserts: the type, number, length of insertions, and positions of interrupted dinucleotide steps in oligonucleotide chain.