Oxidative Stress Impairs the Stimulatory Effect of S100 Proteins on Protein Phosphatase 5 Activity.

Oxidative stress is the consequence of an imbalance between the production of harmful reactive oxygen species and the cellular antioxidant system for neutralization, and it activates multiple intracellular signaling pathways, including apoptosis signal-regulating kinase 1 (ASK1). Protein phosphatase 5 (PP5) is a serine/threonine phosphatase involved in oxidative stress responses. Previously, we reported that S100 proteins activate PP5 in a calcium-dependent manner. S100 proteins belong to a family of small EF-hand calcium-binding proteins involved in many processes such as cell proliferation, differentiation, apoptosis, and inflammation. Therefore, we investigated the effects of oxidative stress on S100 proteins, their interaction with PP5, and PP5 enzyme activity. Recombinant S100A2 was easily air-oxidized or Cu-oxidized, and oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes. The binding of oxidized S100A2 to PP5 was reduced, resulting in decreased PP5 activation in vitro. Oxidation also impaired S100A1, S100A6, S100B, and S100P to activate PP5, although the low dose of oxidized S100 proteins still activated PP5. Hydrogen peroxide (H2O2) induced S100A2 oxidation in human keratinocytes (HaCaT) and human hepatocellular carcinoma (Huh-7) cells. Furthermore, H2O2 reduced the binding of S100A2 to PP5 and decreased PP5 activation in HaCaT and Huh-7 cells. Importantly, even the low dose of S100A2 achieved by knocking down increased dephosphorylation of ASK1 and reduced caspase 3/7 activity in Huh-7 cells treated with H2O2. These results indicate that oxidative stress impairs the ability of S100 proteins to bind and activate PP5, which in turn modulates the ASK1-mediated signaling cascades involved in apoptosis.

Ca2+/S100 proteins inhibit the interaction of FKBP38 with Bcl-2 and Hsp90.

FKBP38 (FK506-binding protein 38), a membrane-anchored TPR (tetratricopeptide repeat)-containing immunophilin, regulates signalling pathways such as cell survival, apoptosis, proliferation and metastasis. However, the mechanisms that regulate the activity of FKBP38 are, at present, poorly understood. We previously reported that Ca2+/S100 proteins directly associate with the TPR proteins, such as Hop [Hsp70 (heat-shock protein of 70 kDa)/Hsp90-organizing protein], kinesin-light chain, Tom70 (translocase of outer mitochondrial membrane 70), FKBP52, CyP40 (cyclophilin 40), CHIP (C-terminus of Hsc70-interacting protein) and PP5 (protein phosphatase 5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore we have hypothesized that Ca2+/S100 proteins can interact with FKBP38 and regulate its function. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, S100B and S100P specifically interact with FKBP38 and inhibit the interaction of FKBP38 with Bcl-2 and Hsp90. Overexpression of permanently active S100P in Huh-7 cells inhibited the interaction of FKBP38 with Bcl-2, resulting in the suppression of Bcl-2 stability. The association of the S100 proteins with FKBP38 provides a Ca2+-dependent regulatory mechanism of the FKBP38-mediated signalling pathways.

S100 proteins modulate protein phosphatase 5 function: a link between CA2+ signal transduction and protein dephosphorylation.

PP5 is a unique member of serine/threonine phosphatases comprising a regulatory tetratricopeptide repeat (TPR) domain and functions in signaling pathways that control many cellular responses. We reported previously that Ca(2+)/S100 proteins directly associate with several TPR-containing proteins and lead to dissociate the interactions of TPR proteins with their client proteins. Here, we identified protein phosphatase 5 (PP5) as a novel target of S100 proteins. In vitro binding studies demonstrated that S100A1, S100A2, S100A6, and S100B proteins specifically interact with PP5-TPR and inhibited the PP5-Hsp90 interaction. In addition, the S100 proteins activate PP5 by using a synthetic phosphopeptide and a physiological protein substrate, Tau. Overexpression of S100A1 in COS-7 cells induced dephosphorylation of Tau. However, S100A1 and permanently active S100P inhibited the apoptosis signal-regulating kinase 1 (ASK1) and PP5 interaction, resulting the inhibition of dephosphorylation of phospho-ASK1 by PP5. The association of the S100 proteins with PP5 provides a Ca(2+)-dependent regulatory mechanism for the phosphorylation status of intracellular proteins through the regulation of PP5 enzymatic activity or PP5-client protein interaction.

Ca(2+) /S100 proteins regulate HCV virus NS5A-FKBP8/FKBP38 interaction and HCV virus RNA replication.

BACKGROUND & AIM: FKBP8/FKBP38 is a unique FK506-binding protein with a C-terminal membrane anchor and localizes at the outer membranes of mitochondria and the endoplasmic reticulum. Similar to some immunophilins, such as FKBP51, FKBP52 and Cyclophilin 40, FKBP8/FKBP38 contain a putative Calmodulin-binding domain and a tetratricopeptide-repeat (TPR) domain for the binding of Hsp90. Both Hsp90 and the non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) interact specifically with FKBP8/FKBP38 through its TPR domain, and the ternary complex formation plays a critical role in HCV RNA replication. The goal of this study is to evaluate that the host factor inhibits the ternary complex formation and the replication of HCV in vitro and in vivo. METHODS: S100 proteins, FKBP38, FKBP8, HCV NS5A, Hsp90, and calmodulin were expressed in E.coli and purified. In vitro binding studies were performed by GST pull-down, S-tag pull-down and surface plasmon resonance analyses. The effect of S100 proteins on HCV replication was analysed by Western blotting using an HCV NS3 antibody following transfection of S100 proteins into the HCV replicon harbouring cell line (sO cells). RESULTS: In vitro binding studies showed that S100A1, S100A2, S100A6, S100B and S100P directly interacted with FKBP8/FKBP38 in a Ca(2+) -dependent manner and inhibited the FKBP8/FKBP38-Hsp90 and FKBP8/FKBP38-NS5A interactions. Furthermore, overexpression of S100A1, S100A2 and S100A6 in sO cells resulted in the efficient inhibition of HCV replication. CONCLUSION: The association of the S100 proteins with FKBP8/FKBP38 provides a novel Ca(2+) -dependent regulatory role in HCV replication through the NS5A-host protein interaction.

Identification and characterization of PRG-1 as a neuronal calmodulin-binding protein.

Intracellular Ca2+-dependent cellular responses are often mediated by the ubiquitous protein CaM (calmodulin), which, upon binding Ca2+, can interact with and alter the function of numerous proteins. In the present study, using a newly developed functional proteomic screen of rat brain extracts, we identified PRG-1 (plasticity-related gene-1) as a novel CaM target. A CaM-overlay and an immunoprecipitation assay revealed that PRG-1 is capable of binding the Ca2+/CaM complex in vitro and in transfected cells. Surface plasmon resonance and zero-length cross-linking showed that the C-terminal putative cytoplasmic domain (residues 466-766) of PRG-1 binds equimolar amounts of CaM in a Ca2+-dependent manner, with a relatively high affinity (a Kd value for Ca2+/CaM of 8 nM). Various PRG-1 mutants indicated that the Ca2+/CaM-binding region of PRG-1 is located between residues Ser554 and Gln588, and that Trp559 and Ile578 potentially anchor PRG-1 to CaM. This is supported by pronounced changes in the fluorescence emission spectrum of Trp559 in the PRG-1 peptide (residues 554-588) upon binding to Ca2+/CaM, showing the stoichiometrical binding of the PRG-1 peptide with Ca2+/CaM. Immunoblot analyses revealed that the PRG-1 protein is abundant in brain, but is weakly expressed in the testes. Immunohistochemical analysis revealed that PRG-1 is highly expressed in forebrain structures and in the cerebellar cortex. Furthermore, PRG-1 localizes at the postsynaptic compartment of excitatory synapses and dendritic shafts of hippocampal neurons, but is not present in presynaptic nerve terminals. The combined observations suggest that PRG-1 may be involved in postsynaptic functions regulated by intracellular Ca2+-signalling.

Identification and characterization of wolframin, the product of the wolfram syndrome gene (WFS1), as a novel calmodulin-binding protein.

To search for calmodulin (CaM) targets, we performed affinity chromatography purification of a rat brain extract using CaM fused with GST as the affinity ligand. Proteomic analysis was then carried out to identify CaM-binding proteins. In addition to identifying 36 known CaM-binding proteins, including CaM kinases, calcineurin, nNOS, the IP(3) receptor, and Ca(2+)-ATPase, we identified an ER transmembrane protein, wolframin [the product of the Wolfram syndrome gene (WFS1)] as interacting. A CaM overlay and an immunoprecipitation assay revealed that wolframin is capable of binding the Ca(2+)/CaM complex in vitro and in transfected cells. Surface plasmon resonance analysis and zero-length cross-linking showed that the N-terminal cytoplasmic domain (residues 2-285) of wolframin binds to an equimolar unit of CaM in a Ca(2+)-dependent manner with a K(D) for CaM of 0.15 muM. Various truncation and deletion mutants showed that the Ca(2+)/CaM binding region in wolframin is located from Glu90 to Trp186. Furthermore, we demonstrated that three mutations (Ala127Thr, Ala134Thr, and Arg178Pro) associated with Wolfram syndrome completely abolished CaM binding of wolframin. This observation may indicate that CaM binding is important for wolframin function and that impairment of this interaction by mutation contributes to the pathology seen in Wolfram syndrome.

Interaction of S100A6 with Target Proteins In Vitro and in Living Cells.

S100A6 is a member of the EF-hand Ca2+-binding protein family, which plays important roles in a wide variety of Ca2+ signaling in the cells, as well as in pathophysiological conditions. Herein, we describe analytical protocols for evaluating the interaction of S100A6 with multiple target proteins in vitro, including biotinylated S100A6 overlay, glutathione-S-transferase (GST)-precipitation, surface plasmon resonance, and a GST-precipitation assay in living cells. These methods will elucidate the detailed molecular mechanisms of S100A6/target interactions and further improve our understanding of the physiological significance of S100A6-mediated Ca2+ signaling. Moreover, they may be used to evaluate other physical S100/target interactions.

Oxidized S100A4 inhibits the activation of protein phosphatase 5 through S100A1 in MKN­45 gastric carcinoma cells.

S100 proteins bind to numerous target proteins, as well as other S100 proteins and activate signaling cascades. S100 proteins can be modified by various post-translational modifications, such as phosphorylation, methylation and acetylation. In addition, oxidation is important for modulating their activities. Previous studies have shown that S100A1 interacts with S100A4 in vitro and in vivo. Due to this potential cross­talk among the S100 proteins, the aim of the present study was to examine whether S100A4 modulates the activity of S100A1. S100A4 was readily oxidized and formed disulfide-linked dimers and oligomers. Although non-oxidized S100A4 bound to protein phosphatase 5 (PP5), the Cu-oxidized S100A4 failed to bind PP5. Instead, the Cu-oxidized S100A4 directly interacted with S100A1 and prevented PP5 activation. Hydrogen peroxide induced S100A4 oxidation in MKN-45 gastric adenocarcinoma cells and decreased S100A1­PP5 interaction, resulted in the inhibition of PP5 activation by S100A1. These data indicate that oxidized S100A4 regulates PP5 activity in a unique manner under oxidative stress conditions.