Cell signalling by reactive lipid species: new concepts and molecular mechanisms

Abstract

The process of lipid peroxidation is widespread in biology and is mediated through both enzymatic and non-enzymatic pathways. A significant proportion of the oxidized lipid products are electrophilic in nature, the RLS (reactive lipid species), and react with cellular nucleophiles such as the amino acids cysteine, lysine and histidine. Cell signalling by electrophiles appears to be limited to the modification of cysteine residues in proteins, whereas non-specific toxic effects involve modification of other nucleophiles. RLS have been found to participate in several physiological pathways including resolution of inflammation, cell death and induction of cellular antioxidants through the modification of specific signalling proteins. The covalent modification of proteins endows some unique features to this signalling mechanism which we have termed the 'covalent advantage'. For example, covalent modification of signalling proteins allows for the accumulation of a signal over time. The activation of cell signalling pathways by electrophiles is hierarchical and depends on a complex interaction of factors such as the intrinsic chemical reactivity of the electrophile, the intracellular domain to which it is exposed and steric factors. This introduces the concept of electrophilic signalling domains in which the production of the lipid electrophile is in close proximity to the thiol-containing signalling protein. In addition, we propose that the role of glutathione and associated enzymes is to insulate the signalling domain from uncontrolled electrophilic stress. The persistence of the signal is in turn regulated by the proteasomal pathway which may itself be subject to redox regulation by RLS. Cell death mediated by RLS is associated with bioenergetic dysfunction, and the damaged proteins are probably removed by the lysosome-autophagy pathway.

Figure 1. Formation of lipid electrophiles via non-enzymatic and enzymatic lipid peroxidation

(A) Arachidonic acid can be converted into several products through enzymatic and non-enzymatic lipid peroxidation. Both free-radical-catalysed as well as enzymatically controlled oxidation yields a subset of products that are electrophilic. 5-HPETE, 5-hydroperoxyeicosatetraenoic acid; LOOH, linoleic acid hydroperoxide. (B) Examples of RLS produced from arachidonic acid and their structures. For simplicity, stereochemistry is not indicated. TXA₂, thromboxane A2; *reactive site.

Figure 2. Classical receptor-mediated signalling compared with signalling mediated through covalent modification

(A) Classical receptor-mediated signalling only occurs when a ligand is present at concentrations which exceed the affinity constant (K_m). This binding is reversible and quickly dissipates when ligand concentrations dip below the needed threshold. (B) Signalling via covalent modification can occur at low concentrations as well as high concentrations of the ligand. Low concentrations of electrophile accumulate over time, resulting in persistent signalling. Higher concentrations of electrophile may result in modification of more diverse protein targets and thus change the cellular response.

Figure 3. RLS differ in their reactivity with specific amino acids

(A) Basic structure of an electrophilic lipid and cylopentenone. The β-carbon of the α,β-unsaturated carbonyl is electrophilic, making the compound reactive with the nucleophilic amino acids cysteine, lysine and histidine. (B) The specificity and reactivity of lipid electrophiles differ depending on relative hardness. Although soft electrophiles such as the cyclopentenones 15d-PGJ₂ and isoprostane J₂ are largely reactive with cysteine residues, harder electrophiles including the isoketals show less specificity and react with many other nucleophilic targets.

Figure 4. Factors which determine susceptibility to thiol modification and cellular thiol targets

Thiol residues have different susceptibilities to being modified by thiol-reactive agents. One important factor is accessibility (within the cell as within a protein) and the pK_a value of the thiol target. (A) The pH of the environment is different, depending on the subcellular location. As shown, the average pH of the mitochondrial matrix is 8–8.5, whereas lysosomes are much more acidic, averaging a pH less than 5.5. The pH, along with the thiol pK_a value determines whether a thiol is deprotonated to form thiolate anion. (B) The local protein environment is a very important determinant of thiol reactivity. For example, an inaccessible, high pK_a protein thiol would be considered the least prone to modification. However, a low pK_a accessible thiol would be a highly sensitive target.

Figure 5. Modification of protein targets involved in the adaptive response

(A) Modification of Keap1 leads to the release of the transcription factor Nrf2 and its translocation to the nucleus. Upon binding to the EpRE, several genes are up-regulated including HO-1, GCL (glutamate-cysteine ligase), GST and NQO1 (NADPH-quinone oxidoreductase). (B) The heat-shock response is also regulated by RLS. Normally present in monomeric form and bound to Hsp70 or Hsp90, HSF1 trimerizes upon exposure to several electrophilic lipids and up-regulates the expression of Hsps by binding to the heat-shock element (HSE). Ub, ubiquitin.

Figure 6. Subcellular localization of protein targets governs the biological response to RLS

The diverse biological effects of electrophilic lipids is due, in part, to their subcellular targets. On the left-hand side, cytosolic targets predominate with the modification of proteins such as Keap1 and the Hsp70/Hsp90, leading to an increase in adaptive responses. On the right-hand side, mitochondrial targets control the response, leading to changes in cellular respiration and apoptotic cell death. In addition, RLS may mediate autophagy and/or mitophagy, leading to proteasome-independent degradation of adducted proteins. ETC, electron-transport chain; HSE, heat-shock element.