Interaction of nucleoredoxin with protein phosphatase 2A

A trimeric protein phosphatase 2A (PP2A(T55)) composed of the catalytic (PP2Ac), structural (PR65/A), and regulatory (PR55/B) subunits was isolated from rabbit skeletal muscle by thiophosphorylase affinity chromatography, and contained two additional proteins of 54 and 55 kDa, respectively. The 54 kDa protein was identified as eukaryotic translation termination factor 1 (eRF1) and as a PP2A interacting protein. The 55 kDa protein is now identified as nucleoredoxin (NRX). The formation of a complex between GST-NRX, PP2A(C) and PP2A(D) was demonstrated by pull-down experiments with purified forms of PP2A, and by immunoprecipitation of HA-tagged NRX expressed in HEK293 cells complexed endogenous PP2A subunits. Analysis of PP2A activity in the presence of GST-NRX showed that NRX competed with polycations for both stimulatory and inhibitory effects on different forms of PP2A.     

Cellular stability of Rho-GTPases glucosylated by Clostridium difficile toxin B

Mono-glucosylation of Rho, Rac, and Cdc42 by Clostridium difficile toxin B (TcdB) induces changes of actin dynamics and apoptosis. When fibroblasts were treated with TcdB, an apparent decrease of the cellular Rac1 level was observed when applying anti-Rac1(Mab 102). This decrease was not based on degradation as inhibition of the proteasome by lactacystin did not stabilise cellular Rac1 levels. The application of anti-Rac1 (Mab 23A8) showed that the cellular Rac1 level slightly increased in TcdB-treated fibroblasts; thus, the apparent loss of cellular Rac1 was not due to degradation but due to impaired recognition of glucosylated Rac1 by anti-Rac1 (Mab 102). In contrast, recognition of RhoA by anti-RhoA (Mab 26C4) and Cdc42 by anti-Cdc42 (Mab 44) was not altered by glucosylation; a transient decrease of cellular RhoA and Cdc42 in TcdB-treated fibroblasts was indeed due to proteasomal degradation, as inhibition of the proteasome by lactacystin stabilised both cellular RhoA and Cdc42 levels. The finding that the apparent decrease of Rac1 reflects Rac1 glucosylation offers a valuable tool to determine Rac1 glucosylation.     

Solution structure and dynamics of human metallothionein-3 (MT-3)

Alzheimer's disease is characterized by progressive loss of neurons accompanied by the formation of intraneural neurofibrillary tangles and extracellular amyloid plaques. Human neuronal growth inhibitory factor, classified as metallothionein-3 (MT-3), was found to be related to the neurotrophic activity promoting cortical neuron survival and dendrite outgrowth in the cell culture studies. We have determined the solution structure of the alpha-domain of human MT-3 (residues 32-68) by multinuclear and multidimensional NMR spectroscopy in combination with the molecular dynamic simulated annealing approach. The human MT-3 shows two metal-thiolate clusters, one in the N-terminus (beta-domain) and one in the C-terminus (alpha-domain). The overall fold of the alpha-domain is similar to that of mouse MT-3. However, human MT-3 has a longer loop in the acidic hexapeptide insertion than that of mouse MT-3. Surprisingly, the backbone dynamics of the protein revealed that the beta-domain exhibits similar internal motion to the alpha-domain, although the N-terminal residues are more flexible. Our results may provide useful information for understanding the structure-function relationship of human MT-3.     

Development of glutathione-coupled cantilever for the single-molecule force measurement by scanning force microscopy

The accuracy and the fidelity of a single-molecule force measurement largely rely on how the molecule of interest is attached to the solid substrate surface (bead, cantilever, cover glass and etc.). A site-specific attachment of a protein without affecting its structure and enzymatic function has been a major concern. Here, we established a glutathione-coupled cantilever to which any glutathione S-transferase (GST)-fused proteins can be attached in a desired direction. The rupture force between glutathione and GST was approximately 100 pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin alpha and importin beta.     

Colocalization and identification of interaction sites between IGFBP-3 and GalNAc-T14

GalNAc-T14 was identified as a novel IGFBP-3 binding partner in previous studies. Here, we furtherly confirmed the interaction between them by confocal microscopy, and identified the binding domain and probable interaction sites of GalNAc-T14 with IGFBP-3. The result of subcellular localization indicated that GalNAc-T14 was distributed in the cytosol, whereas IGFBP-3 existed in the cytosol and nucleolus. Confocal analyses demonstrated that IGFBP-3 and GalNAc-T14 colocalized in the cytosol. The result from yeast two hybrid assay showed that the C terminus of GalNAc-T14 (408-552aa) was essential for the interaction between GalNAc-T14 and IGFBP-3, especially Tyr(408), Pro(409), and Glu(410) of GalNAc-T14 may play key roles in the interaction with IGFBP-3. In conclusion, these studies demonstrated that IGFBP-3 and GalNAc-T14 are colocalized in MCF-7 cells and confirmed the interaction between IGFBP-3 and GalNAc-T14. This interaction may play an important role in the functional regulation of IGFBP-3.     

Tributyltin induces oxidative stress and neuronal injury by inhibiting glutathione S-transferase in rat organotypic hippocampal slice cultures

Tributyltin (TBT) has been used as a heat stabilizer, agricultural pesticide and antifouling agents on ships, boats and fish-farming nets; however, the neurotoxicity of TBT has recently become a concern. TBT is suggested to stimulate the generation of reactive oxygen species (ROS) inside cells. The aim of this study was to determine the mechanism of neuronal oxidative injury induced by TBT using rat organotypic hippocampal slice cultures. The treatment of rat hippocampal slices with TBT induced ROS production, lipid peroxidation and cell death. Pretreatment with antioxidants such as superoxide dismutase, catalase or trolox, suppressed the above phenomena induced by TBT, indicating that TBT elicits oxidative stress in hippocampal slices, which causes neuronal cell death. TBT dose-dependently inhibited glutathione S-transferase (GST), but not glutathione peroxidase or glutathione reductase in the cytosol of rat hippocampus. The treatment of hippocampal slices with TBT decreased the GST activity. Pretreatment with reduced glutathione attenuated the reduction of GST activity and cell death induced by TBT, indicating that the decrease in GST activity by TBT is involved in hippocampal cell death. When hippocampal slices were treated with sulforaphane, the expression and activity of GST were increased. Notably, TBT-induced oxidative stress and cell death were significantly suppressed by pretreatment with sulforaphane. These results indicate that GST inhibition could contribute, at least in part, to the neuronal cell death induced by TBT in hippocampal slices. This study is the first report to show the link between neuronal oxidative injury and the GST inhibition elicited by TBT.