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Review
. 2019 May:23:101114.
doi: 10.1016/j.redox.2019.101114. Epub 2019 Jan 16.

Regulation of stress signaling pathways by protein lipoxidation

Tommi Patinen  1 , Simone Adinolfi  1 , Carlos Cruz Cortés  2 , Jouni Härkönen  1 , Ashik Jawahar Deen  1 , Anna-Liisa Levonen  3
Affiliations
Review

Regulation of stress signaling pathways by protein lipoxidation

Tommi Patinen et al. Redox Biol. 2019 May.

Abstract

Enzymatic and non-enzymatic oxidation of unsaturated fatty acids gives rise to reactive species that covalently modify nucleophilic residues within redox sensitive protein sensors in a process called lipoxidation. This triggers adaptive signaling pathways that ultimately lead to increased resistance to stress. In this graphical review, we will provide an overview of pathways affected by protein lipoxidation and the key signaling proteins being altered, focusing on the KEAP1-NRF2 and heat shock response pathways. We review the mechanisms by which lipid peroxidation products can serve as second messengers and evoke cellular responses via covalent modification of key sensors of altered cellular environment, ultimately leading to adaptation to stress.

Keywords: Cysteine modification; HSF1; Heat shock proteins; KEAP1; NRF2; Protein lipoxidation; Redox regulation; Stress response.

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Figures

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Graphical abstract
Fig. 1
Fig. 1
Activation of the KEAP1-NRF2 and HSF1 pathways by electrophilic lipoxidation products. To survive, cells have developed an intricate set of stress signaling pathways that are activated by endogenous or exogenous signals . Central to these defenses are the KEAP1-NRF2 and HSF1 pathways that together regulate hundreds of genes via binding to antioxidant response and heat shock elements, respectively , . A) The main signaling proteins of KEAP1-NRF2 pathway are the transcription factor NRF2 and its negative regulator protein KEAP1, which is a cullin-3 (CUL3)-RING ubiquitin ligase adaptor/scaffold protein enabling rapid proteasomal degradation of NRF2 during unstressed conditions. NRF2 is bound by the BTB domains of the KEAP1 dimer via DLG and ETGE motifs residing in NRF2 Neh2 domain. During the activation by e.g. lipid-derived electrophiles, the proteasomal degradation machinery is disrupted and de novo synthetized NRF2 is free to enter the nucleus to heterodimerize with the members of the musculoaponeurotic fibrosarcoma oncogene homolog protein family (sMAF) and drive the expression of cytoprotective genes . SH; Sulfhydryl group of free cysteine residue, Ub; Ubiquitin. B) The HSF1 pathway consists of inactive HSF1 monomer that is bound by heat shock proteins from HSP90α and HSP70 families. HSF1 is negatively regulated by HSPs . During stress, the interaction is disrupted and HSF1 trimerizes and enters the nucleus to regulate the heat shock response genes . While the two pathways are largely distinct, they converge at the level of shared stimulus (e.g. electrophilic lipid peroxidation products) and mode of action (i.e. modification of redox-active cysteines that are regarded as molecular “switches”) .
Fig. 2
Fig. 2
Cysteine residues are critical mediators of the antioxidant and heat shock responses. A) In the KEAP1-NRF2 pathway, the KEAP1 protein is responsible for sensing oxidant/electrophile stress. Human KEAP1 has 27 cysteines in total, enriched in the IVR-domain , . Cysteine modification leads to conformational changes in KEAP1, resulting in disruption of KEAP1-mediated ubiquitination of NRF2 . B) There are seven cysteine residues in human NRF2, implicated to take part in oxidant/electrophile sensing . However, no studies have addressed their impact on LDE-mediated NRF2 activation. C) Human HSF1 contains five cysteines, two of which (C35 and C135) have been shown to form a dimer upon heat stress or by H2O2, . However, no studies to date have identified LDE targets in HSF1. D) Molecular chaperone family of HSP70s are one of the more abundant HSPs that regulate HSF1 , . HSP70s have five cysteines. E) Human HSP90α family of HSPs contain seven cysteines that are reactive towards heat shock and oxidative stress , .
Fig. 3
Fig. 3
Targets of LDEs in the KEAP1-NRF2 and HSF1 pathways. The ability of LDEs to activate cell signaling pathways depend on their reactivity with sulfhydryl groups. Lipoxidation is a process that does not affect cellular proteins randomly as it is directed by the differences in the binding strength, pKa, type of the Michael acceptors, shape of the electrophilic molecule, and the presence of other amino acids close to the reactive cysteine , , , , , , , , , . Modification of thiol targets elicit downstream transcriptional responses that alleviate oxidant and proteotoxic stress . NRF2 encodes a myriad of antioxidant genes by binding to highly conserved ARE sites within enhancer regions of genes after forming a heterodimer with a small MAF protein in the nucleus . The highly conserved NRF2 target genes providing protection against xenobiotics include NAD(P)H:quinone oxidoreductase 1 (NQO1) , , , , Phase II enzyme Glutathione S-transferase (GST) and Multidrug-resistance-associated proteins (MRPs) . Additionally, NRF2 regulates gene expression of enzymes involved in glutathione metabolism as well as antioxidants. These include glutamate-cysteine ligase catalytic (GCLC) and modifier (GCLM) subunits , thioredoxin reductase 1 (TXNRD1) and antioxidant enzymes from peroxiredoxin family (e.g. PRDX1) , . NRF2 also encodes autophagosome cargo protein sequestome 1 (SQSTM1), which binds other proteins for specific autophagy . Upon activation of the heat shock response, HSF1 forms first an active trimer and activating genes having a HSE regulatory element, such as immediate early response 5 (IER5) and multiple heat shock proteins (chaperones) that have various different functions. These genes include heat shock protein family A (HSP70) member 8 (HSPA8) , Heat shock protein family D/E (HSP60/HSP10) member 1 (HSPD1/E1) , 40-kDa heat shock proteins from HSP40 family (HSP40) . HSF1 can also provide the means to repair already damaged DNA by encoding DNA polymerase kappa (POLK) and through a complex with Poly(ADP-ribose) polymerase 1 and 13 (PARP1 and PARP13) , , . KEAP1-NRF2 and HSF1 pathways co-activate and/or regulate heme oxygenase 1 (HMOX1). Both pathways are thus complementary and overlapping in their functions , , , .
Fig. 4
Fig. 4
Emerging novel methods to identify thiol targets of LDEs. A) In competitive activity-based profiling method with isotope labeling, cells are treated with DMSO or LDE, after which the proteins are labeled with an iodoacetamide alkyne probe and isotopically-labeled, TEV protease-cleavable biotin tags are incorporated with click chemistry. After enrichment with streptavidin, sequential on-bead protease digestion is done to yield probe-labeled peptides for MS analysis. B) T-REX (Targetable reactive electrophiles and oxidants) assisted RES delivery utilizes RES linked signaling molecules to increase on-target signaling output , . Unlike conventional methods of dosing, where the cells are exposed to extracellular RES dosing, T-REX method aims to limit the activation of multiple signaling pathways simultaneously. This is achieved with functional Halo-fusion protein complexes expressed by the live cell, which are activated via photo-uncaging to release the RES . Identification of cysteine targets is performed from the cell lysate using LC-MS. POI; protein of interest. C) G-REX (Genome-Wide reactive electrophiles and oxidants) is a high-throughput version of T-REX that achieves similar results while utilizing cell lines that express only HaloTag . The cells are subsequently treated with specifically designed photocaged probes before MS-analysis of total proteome. D) To identify targets of endogenously produced LDEs, stable isotope labeling with amino acids in cell culture (SILAC) is performed, which is coupled with incorporation of alkyne-labeled linoleic acid (aLA). The cells are subsequently activated with Kdo2-lipidA to produce terminal alkyne labeled RES through lipid peroxidation . After copper-mediated click chemistry, modified proteins are affinity purified and analyzed by MS.
Fig. 5
Fig. 5
Biological consequences of activation of KEAP1-NRF2 and HSF1 pathways by lipid-derived electrophiles. Both KEAP1-NRF2 and HSF1 pathways are modified with diverse set of exo- or endogenous compounds , , , , , , , , , . In the light of maintaining redox-homeostasis, both pathways reduce proteotoxic stress and inflammation. There are evidences of cross-talk between these two pathways by certain compounds and also cross-protection by encoded genes during the course of their diverse signaling mechanisms. Reviewed: . These pathways perform and maintain important cellular functions as well as some key regulatory effects, which make them interesting in the context of health and disease. The KEAP1-NRF2 pathway can modulate apoptosis , metabolic control , mitochondrial biogenesis and critical steps in the development of organisms , , . Like NRF2, HSF1 also has important role in the normal development , , regulation of normal chaperone machinery and glucose metabolism .
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