Redox Regulation of PPARγ in Polarized Macrophages.

Verena Trümper,Andreas Von Knethen,Juliana Heidler,Florian Richter,Bernhard Brüne,Ilka Wittig

doi:10.1155/2020/8253831

Verena Trümper, Andreas Von Knethen + Show 4 more

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https://doi.org/10.1155/2020/8253831

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Abstract

The peroxisome proliferator-activated receptor (PPARγ) is a central mediator of cellular lipid metabolism and immune cell responses during inflammation. This is facilitated by its role as a transcription factor as well as a DNA-independent protein interaction partner. We addressed how the cellular redox milieu in the cytosol and the nucleus of lipopolysaccharide (LPS)/interferon-γ- (IFNγ-) and interleukin-4- (IL4-) polarized macrophages (MΦ) initiates posttranslational modifications of PPARγ, that in turn alter its protein function. Using the redox-sensitive GFP2 (roGFP2), we validated oxidizing and reducing conditions following classical and alternative activation of MΦ, while the redox status of PPARγ was determined via mass spectrometry. Cysteine residues located in the zinc finger regions (amino acid fragments AA 90-115, AA 116-130, and AA 160-167) of PPARγ were highly oxidized, accompanied by phosphorylation of serine 82 in response to LPS/IFNγ, whereas IL4-stimulation provoked minor serine 82 phosphorylation and less cysteine oxidation, favoring a reductive milieu. Mutating these cysteines to alanine to mimic a redox modification decreased PPARγ-dependent reporter gene transactivation supporting a functional shift of PPARγ associated with the MΦ phenotype. These data suggest distinct mechanisms for regulating PPARγ function based on the redox state of MΦ.

Highlights

Reactive oxygen species (ROS) fulfill important mediator functions in a variety of cellular signaling pathways regulating aspects of inflammation, cell proliferation and differentiation, apoptosis, iron homeostasis, and defense mechanisms [1,2,3]
Redox-based posttranslational modifications (PTMs) of redox-sensitive GFP2 (roGFP2) changed the spectral properties of the fluorophore, which could be calculated by relative fluorescence intensities (RFI) at the ratio of 405/488 nm (Figure 1)
M(LPS/interferon γ (IFNγ)) polarization was characterized by a significant, exponential increase of oxidizing conditions in the cells indicated by a relative fluorescence intensity (RFI) of 1.02 (±0.02) after 1 h, 1.04 (±0.01) at 2 h, and 3.26 (±0.3) at 48 h

Summary

Introduction

Reactive oxygen species (ROS) fulfill important mediator functions in a variety of cellular signaling pathways regulating aspects of inflammation, cell proliferation and differentiation, apoptosis, iron homeostasis, and defense mechanisms [1,2,3]. ROS trigger redox-based posttranslational modifications (PTMs) changing protein folding and stability. The efficiency of these chemical reactions depends on different criteria including rate constants between oxidants and amino acids, residue accessibility, its pKa, and, the properties of neighboring amino acids [4]. Nucleophilic thiolates can be catalytically active, and the connection of the cysteine residues with other functional residues ensures special properties of proteins. Alterations of their chemical behavior drastically change the function of the whole protein. One example is the peroxisome proliferator-activated receptor gamma (PPARγ)

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