Cytochrome c takes on plasmalogen catabolism

Abstract

Plasmalogens—phospholipids containing a characteristic vinyl ether group—are precursors of lipids important for cellular signaling such as arachidonic acid. Plasmalogen catabolism involves cleavage of the vinyl ether bond, but the identity of the corresponding enzyme that cleaves the sn-1 vinyl ether bond was unknown. New research shows that cytochrome c, with some help from another lipid, catalyzes the oxidative cleavage of this bond. This discovery, and the subsequent mechanistic dissection, provides exciting new directions for lipid signaling research.

Introduction

Plasmalogens are a type of phospholipid defined by the presence of an alkene group next to an ether linkage (Fig. 1). They evolved early in the history of life on earth. Plasmalogens are found in animals, as well as many anaerobic bacteria, but not in fungi, plants, or bacteria with respiratory systems that produce ROS,² which can react with and destroy them (1).

Figure 1.

Plasmalogenase activity and its consequences. Proposed pathway by which cyt c activated by cardiolipin (cyt c*) and ROS hydrolyzes plasmalogens to produce α-hydroxyaldehydes and a lysophospholipid; these lipids can be further hydrolyzed by lysophospholipase to carboxylic acids like arachidonic acid, the precursor of the eicosanoids. The oxygen atoms in the released α-hydroxy group are color-coded to match the incorporated substrate. The vinyl ether group is highlighted in gold. X is ethanolamine or choline.

Plasmalogens are abundant in the central nervous systems of animals, suggesting they may play roles related to nerve transmission. However, their presence in single-cell animals, which do not have a nervous system, suggests that they have also evolved to fulfill other functions, as units of membrane structure and as participants in signaling.

Sensitivity to ROS has also led to the proposal that plasmalogens could serve as protection against ROS; in this model, hydrolysis by an unknown ROS-consuming mechanism could be reversed by a well-known acyl-CoA–dependent reaction (2).

The pathway by which plasmalogens are produced in animal cells is known: An oxygen-dependent desaturation creates the characteristic double bond from a saturated substrate (1). The pathway by which plasmalogens are degraded, on the other hand, has been a mystery since their discovery in 1924. Solving this mystery is of more than academic interest, because one of the typical metabolites of this degradation is arachidonic acid, which can modulate ion channel kinetics (3) and is the precursor to the eicosanoids, with their multiple and diverse signaling functions, including the regulation of inflammation, fever, pain, and blood-clotting (4). It is known that two cleavages must occur, one at the vinyl ether group (the sn-1 chain of the glycerol backbone) and another to release the sn-2 chain; the identity of an enzyme that cleaves the vinyl ether linkage has remained obscure. In this issue of JBC, Jenkins et al. (3) report the surprising discovery that cytochrome c (cyt c), with help from an activating lipid and appropriate oxidants, performs this reaction. This unexpected finding, including a new enzymatic mechanism, offers a host of new questions for scientists across a variety of disciplines.

Cyt c is best known for its role in transferring electrons from complex III to complex IV, while bound to the inner membrane of the mitochondrion. However, cyt c can also function as a peroxidase, which is induced by the binding of the mitochondrial lipid cardiolipin (CL), resulting in a change in the conformation of the protein and a dramatic change in its redox potential from +260 to −400 mV. This abrogates its electron transfer function and shifts its activity to that of a peroxidase. Earlier work showed that activated cyt c can oxidize polyunsaturated linoleoyl-acyl chains in CL (5); although these chains do not contain the vinyl ether group, the unsaturated bonds and overall framework were sufficiently similar to that of plasmalogens to lead Jenkins et al. (3) to consider the possibility that activated cyt c could be the missing hydrolytic enzyme.

This hypothesis was verified by testing the normal peroxidase conditions for cyt c—addition of CL and H₂O₂—and observing plasmalogen turnover. The authors then conducted a series of elegant experiments based on high resolution, high mass accuracy (HRAM) MS, compound derivatization, and stable isotopes to further validate these findings and investigate the catalytic mechanism. Using ¹⁸O-labeled O₂, H₂O, and H₂O₂, followed by MS of the reaction products, the authors show that oxygen from O₂ is incorporated into the hydroxyl group, and oxygen from H₂O is found at the aldehyde carbon of the resulting α-hydroxy long-chain aldehydes (Fig. 1). This differs from normal peroxidase mechanisms in that neither of the H₂O₂ oxygen atoms is incorporated into the product. Of great interest is the finding that the peroxidase activity of cyt c can be activated by oxidized CL in the absence of H₂O_2. Other negatively charged lipids such as PI(4,5)P₂ and PI(3,4,5)P₃ were also able to activate cyt c plasmalogenase activity. The reaction could be inhibited by a mAb against cyt c, as well as known inhibitors of cyt c's peroxidase activity, cyanide and azide, confirming the role of the protein in the reaction (6).

The authors place these results in the context of damage induced by oxidative stress to the myocardium, which is enriched in both mitochondria and plasmalogens. During infarction/reperfusion, mitochondria close to the sarcoplasmic reticulum (SR)—a specialized type of ER found in muscle cells—can release cyt c, which can act on AA-rich plasmalogens in the SR. A similar process could take place at the muscle cell membrane. They also note that a number of studies have shown significant decreases in plasmalogens in the brains of Alzheimer's disease patients as well as accumulation of α-hydroxyaldehyde, which correlates with the severity of the disease (7). Thus, mitochondrial dysfunction may lay the groundwork for ensuing dementia. Recent animal and clinical studies provide further impetus for this mechanism, showing modest effects of plasmalogen administration on cognitive function in a rat model of AD and in female patients with mild cognitive impairment (8, 9). The research by Jenkins et al. (3) therefore offers new entry points to modulating these processes.

Since activated cyt c promotes oxidation of polyunsaturated fatty acids in CL (3) and oxidized CL activates hydrolysis of plasmalogens in the presence of O₂, a cascade through oxidized CL can lead to the release of AA-rich lysolipids, AA, and the downstream cascade of eicosanoid production. Now further interesting questions arise: The authors suggest that oxidized CL hydroperoxides replace H₂O₂ in the observed plasmalogenase activity. The mechanism by which oxidized CL accomplishes this task by itself will be of considerable interest. Do other plasmalogenases exist, using the same chemical mechanism or others, or is cyt c the only one? How does activated cyt c catalyze this reaction? Does this new, critical function prompt a reevaluation of what we know about cytochrome c's basic biology? It seems the resolution of one mystery has created many more.