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Fuminori Yamaguchi1, Mitsumasa Tsuchiya, Seiko Shimamoto
1Department of Cell Physiology, Faculty of Medicine, Kagawa University.
Abstract:
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.
Insights
Oxidative stress impairs S100 proteins' ability to activate Protein Phosphatase 5 (PP5). This reduced PP5 activity affects the apoptosis signal-regulating kinase 1 (ASK1) pathway, impacting cell death signaling.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Signaling
Background:
- Oxidative stress disrupts cellular redox balance, activating signaling pathways like apoptosis signal-regulating kinase 1 (ASK1).
- Protein phosphatase 5 (PP5) is a key enzyme in oxidative stress response, regulated by S100 proteins.
- S100 proteins are calcium-binding proteins involved in various cellular processes, including apoptosis.
Purpose of the Study:
- To investigate the impact of oxidative stress on S100 protein interaction with PP5.
- To determine how oxidative stress affects PP5 enzyme activity.
- To elucidate the role of S100-PP5 interaction in modulating ASK1 signaling under oxidative stress.
Main Methods:
- Oxidation of recombinant S100A2 and other S100 proteins using air or copper.
- Assessing S100 protein binding to PP5 and PP5 activation in vitro.
- Inducing oxidative stress with hydrogen peroxide (H2O2) in keratinocyte (HaCaT) and hepatocellular carcinoma (Huh-7) cell lines.
- Measuring S100A2 oxidation, S100A2-PP5 binding, and PP5 activity in cells.
- Evaluating ASK1 dephosphorylation and caspase 3/7 activity following S100A2 knockdown in H2O2-treated Huh-7 cells.
Main Results:
- Oxidized S100A2 formed cross-linked dimers and higher molecular-mass complexes.
- Oxidation reduced the binding of S100A2 to PP5, leading to decreased PP5 activation in vitro.
- Oxidative stress impaired the ability of various S100 proteins (S100A1, S100A6, S100B, S100P) to activate PP5.
- H2O2 induced S100A2 oxidation and reduced S100A2-PP5 binding and PP5 activation in HaCaT and Huh-7 cells.
- S100A2 knockdown in Huh-7 cells promoted ASK1 dephosphorylation and reduced caspase 3/7 activity under H2O2 treatment.
Conclusions:
- Oxidative stress impairs the interaction between S100 proteins and PP5, diminishing PP5's enzymatic activity.
- This impairment affects the ASK1-mediated signaling pathway, influencing apoptotic cascades.
- S100 proteins play a crucial role in regulating cellular response to oxidative stress via PP5 modulation.
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