The electrophile responsive proteome: integrating proteomics and lipidomics with cellular function

Abstract

Significance: The process of lipid peroxidation is emerging as an important mechanism that mediates the post-translational modification of proteins. Through advanced analytical techniques, lipidomics is now emerging as a critical factor in our understanding of the pathology of a broad range of diseases.

Recent advances: During enzymatic or nonenzymatic lipid peroxidation, the simple structure of an unsaturated fatty acid is converted to an oxylipidome, many members of which are electrophilic and form the reactive lipid species (RLS). This aspect of lipid biology is particularly important, as it directly connects lipidomics with proteomics through the post-translational modification of a sub-proteome in the cell. This arises, because the electrophilic members of the oxylipidome react with proteins at nucleophilic amino-acid residues and so change their structure and function to form electrophile-responsive proteomes (ERP).

Critical issues: Biological systems have relatively few but well-defined and mechanistically distinct pro-oxidant pathways generating RLS. Defining the ERPs and the mechanisms underlying their formation and action has been a major focus for the field of lipidomics and redox signaling.

Future directions: We propose that a unique oxylipidome can be defined for specific oxidants and will predict the biological responses through the reaction with proteins to form a specific ERP. In this review, we will describe the ERPs that modulate antioxidant and anti-inflammatory protective pathways, including the activation of Keap1/Nrf2 and the promotion of cell death through interactions with mitochondria.

FIG. 1.

Oxylipidomics: the formation of structurally diverse reactive lipid species (RLS). (A) As can be seen in this Venn diagram, the lipid peroxidation products formed depend on the method of initiation. The black and white circles represent the oxylipidomes produced by nonenzymatic and enzymatic lipid peroxidation, respectively. While enzymatic lipid peroxidation results in a relatively small oxylipidome with a few key products, nonenzymatic lipid peroxidation results in a larger, less specific oxylipidome. Some products, including malondialdehyde, are common to both enzymatic and nonenzymatic lipid peroxidation (shown as gray overlap). (B) Lipid peroxidation can be initiated by a number of diverse mechanisms such as through enzymes, including cyclo-oxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP). Nonenzymatic stimuli for lipid peroxidation include peroxynitrite (ONOO−, which in acid form breaks down to the radicals NO₂^• and ^•OH), hydroxyl radical (^•OH), lipid alkoxyl radical (LO^•), lipid peroxyl radical (LOO^•), and myoglobin (Mb) or hemoglobin (Hb), which form ferryl radical species (Mb^•/Hb^•).

FIG. 2.

Products of enzymatic and nonenzymatic lipid peroxidation. Products of enzymatic and nonenzymatic lipid peroxidation are different. While enzymatic lipid peroxidation forms electrophilic species (*denotes reactive carbon) such as the cyclopentenone prostaglandins as well as electrophilic fatty acids (EFOX), nonenzymatic lipid peroxidation results in the formation of a more diverse set of electrophiles including, but not limited to, small aldehydes, isoketals, and isoprostanes. Structures shown are examples of products formed during these two types of lipid peroxidation.

FIG. 3.

The effects of lipid peroxidation on cell function. Both positive and negative effects of lipid peroxidation products have been reported in the literature. During pathology, nonenzymatic sources of lipid peroxidation, including various oxidants and heme proteins, can increase the RLS involved in damaging cellular proteins, depleting antioxidants (e.g., glutathione), and inducing mitochondrial dysfunction. The net effect is toxicity and death of the cell. On the other hand, physiological lipid peroxidation occurs mainly through enzymatically controlled mechanisms and can be involved in the resolution of inflammation (through modification of inhibitor of nuclear factor kappa-B kinase subunit beta, for example), increase in cellular antioxidants through nuclear factor-erythroid 2 related factor (Nrf2)/Keap1, and cytoprotection. The overall effect of these physiological mediators is the preservation of normal tissue and protection from secondary stressors.

FIG. 4.

Modification of protein targets determines whether cellular response leads to physiological changes or pathology. The proteomes modified by RLS determines cellular fate. Two examples are the modification of Keap1, which leads to increased antioxidant production (a protective mechanism) and the modification of the 20S proteasome, leading to the inhibition of proteasomal activity (detrimental effect). (A) Usually, Keap1 and Nrf2 are bound in the cytosol. However, on oxidation of key thiol residues on Keap1 by electrophiles or other species, Keap1 releases Nrf2. The transcription factor, Nrf2 then translocates to the nucleus and binds the electrophile response element (EpRE), leading to the transcription of genes involved in antioxidant defenses within the cell. (B) Usually, the 20S proteasome is responsible for degrading oxidized proteins to peptides that can be used within the cell for new protein synthesis. However, on modification of key sites, the chymotryptic and tryptic activity of the proteasome is inhibited and leads to decreased activity of the proteasome. Proteasomal inhibition results in the build-up of proteins usually targeted to this degradation pathway, which can contribute to pathology.

FIG. 5.

The proteomes modified by oxylipidome. (A) Lipid peroxidation can be initiated by a number of diverse mechanisms such as Mb, inducible nitric oxide synthase (iNOS), and COX, as shown here. Since each initiating mechanism has distinct attributes, this results in a family of oxylipidomes (a–c). In a specific cell type, the oxylipidome then interacts with the nucleophilic proteome to generate a cell and oxylipidome-specific electrophile responsive proteome represented as the spot patterns on 2D gels. (B) Just as the RLS produced are diverse in nature (Fig. 1), the proteomes modified differ depending on physiological and pathological stressors, here represented as a Venn diagram. Importantly, while soft electrophiles produced in physiology are more likely to react with soft nucleophiles such as cysteine, more reactive species are produced by free-radical oxidation of polyunsaturated fatty acids in disease. These highly reactive species can react with other nucleophilic amino acids, including histidine (His) and lysine. The net effect on cell function depends on the specificity of the modification. While the modification of cysteine residues can lead to redox signaling as well as sometimes detrimental effects (e.g., if Cys is in the active site of an enzyme), the modification of lysine and histidine residues primarily causes protein damage, leading to events such as bioenergetic dysfunction, inflammation, protein aggregation (due to crosslinks), and inhibition of the proteasome.