Ferroptosis - part 2: more death by iron

PHAROAH-TOES-ISSSS!!!!

Ahem, I mean, ferroptosis. I haven’t quite finished this topic. Heck, I’ve barely even begun as I’ve found out. Going through my to-be-read-someday folder of papers that I gradually gather over time (like I’m sure most people who work in the sciences have), I came across a News & Reviews article talking about ferroptosis by D’Herde & Krysko (2017). This wasn’t cited in the previous review I was reading from the last post by Liang et al. (2022), so I’m not sure where I got the article. I’m thinking it might have been a stray Google Scholar search idly looking up anything on the subject. But it laid out much of what I didn’t pick up from the first review. Specifically that ferroptosis is driven by very specific lipid molecular species and that their synthesis is driven by a specific enzyme.

In my last post I may have given the impression that there’s a whole bunch of oxidation of lipids going on during ferroptosis all wily-nily. And while there is oxidation of many lipid molecular species going on, the interesting research article by Kagan et al. (2017), as discussed by D’Herde & Krysko (2017), points out that the induction of ferroptosis can be pinned down to two lipid molecular species, which are phosphatidylethanolamines (PE) that have fatty acid compositions of either 18:0/20:4 (stearic acid/arachidonic acid) or 18:0/22:4 (stearic acid/adrenic acid). That was something I had not expected. In my mind, as I was reading on this subject, I was thinking something more along the lines of a general degradation of lipids that would ultimately lead to death. But it seems, as is usual in the case of biological life, that it’s a little more complicated than that. And in other ways, much more specific. Below are figures showing the chemical structures of PEs with the specified fatty acids (Fig. 1):

Figure 1. Phosphatidylethanolamines (PE) that are oxygenated during ferroptosis and signals for cell death. A) PE 18:0/20:4 and B) PE 18:0/22:4

PEs are a lipid class I feel are sometimes overlooked or forgotten. They’re there but it’s usually their cousin, phosphatidylcholines (PC), that tend to take center stage. PC are often the lipids students will learn about in their general cell biology or biochemistry classes rather than the whole suite and complicated mixture of lipids that make up the cell membrane. As the current model of the cell membrane is a bilayer, each side, which are called ‘leaflets’, can have a different composition of lipids. The ‘outer leaflet’, i.e. the side of the cell membrane that is the outside of the cell, contains many of the PC molecular species. Also there are sphingolipids, cholesterol (in mammalian cells; other types of sterols in other organisms), and others, especially those that associate with the extracellular matrix like glycolipids. Within the ‘inner leaflet’, i.e. the face of the cell membrane that is inside the cell, there are the PE, but also phosphatidylserine, phosphatidylinositols (a future post on those is coming), and other lipids. As cell death, such as ferroptosis, is concerned with the inner workings of the cell, it would make sense that the signal would ‘come from inside the house’, so to speak. Below is a crude cartoon (don’t take it to be accurate or representative of the locations or proportions of the shown lipids) showing a model of a cell membrane, representing lipids in their ‘ball and squiggly line’ form (Fig. 2). The ‘balls’ in this case are the ‘phospho-’ heads, while the two squiggly lines represent the two fatty acids that are attached to phosphoglycerol lipids that make up cell membranes.

Figure 2. Cartoon of a cell membrane, depicting the outer and inner leaflets.

In the article by Kagan et al. (2017), they used an inhibitor to the enzyme glutathione peroxidase 4 (GPX4), the enzyme I mentioned in the previous post. This enzyme uses glutathione, a powerful redox cofactor (i.e. a molecule capable of accepting and giving electrons) to reduce oxidatively damaged phospholipids. Specifically it acts on the fatty acids of phospholipids that have had a hydroperoxy group added to them. This is a functional group of two oxygens bonded together by a single bond with a hydrogen on its outer end (see Fig. 3 below):

Figure 3. Hydroperoxide on a fatty acid.

GPX4 will convert the hydroperoxy group into a relatively benign hydroxl group (i.e. removing one of the oxygens as depicted in Fig. 3). From there the lipid may carry on with its business, or another enzyme can come along to exchange the hydroxy fatty acid for a nonhydroxy fatty acid. It turns out that GPX4 is a necessary enzyme for life since its deletion is lethal. So the mitigation of ferroptosis is an important process, as much as ferroptosis may be an important process in of itself. Inhibiting the action of GPX4, as the authors of the research had done, they created a system to test for what lipids would be signals of ferroptosis. Using mass spectrometric analysis of the lipids following treatment with the GPX4 inhibitor, as well as on cells that were GPX4 knockouts, they found a little over a hundred phospholipid species that were oxygenated in some way. They say they found oxygenated species in each of the phospholipid classes that they measured with the notable exception of cardiolipin. This lipid class is found in the mitochondria, and its oxygenation is usually a herald of apoptosis, a form of cell death, but is untouched in the ferroptosis type of cell death, making the two distinct cellular processes. The authors of the study filtered their data further, selecting against the fold increases and significance in those changes, to determine if specific lipids would indicate a ferroptosis cell death signal. These selection criteria narrowed down their hundred lipids species down to four. These were the oxygenated PE species containing arachidonic or adrenic acids (Fig. 1; note that the structures in Fig. 1 aren’t shown as oxygenated).

What I thought was quite interesting, besides the fact that they could narrow down the cell death signals to such specific molecular species, was that the ones they found contained arachidonic and adrenic acids. In other cellular processes, arachidonic acid and adrenic acids are converted to what are called ‘lipid mediators’, which are hydroxylated polyunsaturated fatty acids that act as signaling molecules, especially in the immune system. Usually the polyunsaturated fatty acids are first cleaved off the phospholipids due to the enzyme phospholipase A. The free fatty acid then can become oxygenated from a variety of enzymes, one class of which is known as lipoxygenases (LOX). These enzymes are designated by a number for where in the fatty acid chain they add a hydroxyl group. For example, 15-LOX adds a hydroxyl group to the 15^th position of arachidonic acid (or adrenic acid) counting from the carboxylic acid end. In fact, it was 15-LOX that Kagan et al. (2017) found to be responsible for oxygenating the specific PE molecular species that they found; no other enzymes that they tested were implicated. They found this by testing a variety of inhibitors that targeted 15-LOX as well as other oxygenating enzymes within cells treated with the GPX4 inhibitor (that which would induce ferroptosis). When adding the 15-LOX inhibitors they found less ferroptosis cell death in the presence of the GPX4 inhibitor, suggesting a sort of rescue effect due to the lowered amount of oxygenated PE species. Even more interesting was that they found that 15-LOX didn’t act on the free fatty acid to elicit ferroptosis, but rather acted on the fully formed PE molecular species itself. They added oxygenated fatty acids to the cells treated with the GPX4 inhibitor, but this didn’t increase ferroptosis cell death relative to their control. But when they added oxygenated PE, they found it would increase ferroptosis cell death. So there’s something rather particular about the oxygenated PE molecular species and not just its fatty acid parts.

This connection between ferroptosis, 15-LOX, and oxygenated arachidonic and adrenic acids on PE, makes me wonder what sort of connection all this might have in other signaling pathways. Is ferroptosis a source of signaling molecules that might recruit other cells, such as immune cells, that would respond to what seems to be trouble? In this scenario the oxygenated fatty acid would need to be cleaved off and actually leave the cell. Although if ferroptosis leads to cell death, I would imagine that the cellular contents would eventually spill out. And if the ferroptosis is something widespread, in more than just a single cell, then this could indicate a wider systemic problem that would require the immune system to respond. Another question that comes to my mind is given that the other oxygenating enzymes were not involved, why is 15-LOX so special to be involved with ferroptosis? I’ll have to do more reading on this.

Now, there’s one more enzyme that’s directly involved I haven’t yet talked about, which is acyl-CoA synthase 4 (ACSL4). Kagan et al. (2017) used a knockout of this enzyme throughout their research, but the significance of ACSL4 is greatly explained in the accompanying research article by Doll et al. (2017). Here, the authors used two different screens to find genes that would show resistance to ferroptosis. In both of those screens ACSL4 popped up, resisting ferroptosis even when GPX4 was knocked out. The enzyme ACSL4 is responsible for producing acyl-CoA. These are substrates to other enzymes that add fatty acids to glycerol lipids, such as phospholipids, including PE. In particular, ACSL4 has a preference for very long chain polyunsaturated fatty acids, such as arachidonic and adrenic acids. So, by knocking out ACSL4 so that it is no longer active would lead to less arachidonyl-CoA and adrenyl-CoA, and less of that substrate means that it cannot be incorporated on to phospholipids. Without those particular fatty acids on phospholipids, and, according to Kagan et al. (2017), PE especially, they are not available to become oxygenated when reactive oxygen species are present. This prevents the formation of those hydroperoxy PE species that Kagan et al. (2017) found to be indicators of cell death by ferroptosis.

Another interesting finding within the Doll et al. (2017) research is that ACSL4 expression is correlated with more aggressive cancers. At first this seemed counterintuitive because it seemed that this would mean they should be more susceptible to ferroptosis. And perhaps that would be the case assuming their GPX4 had lower expression, or other enzymes that mediate reactive oxygen species were less expressed. But if that’s not the case, then the cancer cells may thrive unimpeded from ferroptotic cell death. If, however, drugs were used to induce ferroptosis (e.g. such as by inhibiting GPX4), then that higher expression of ACSL4 may work against such aggressive cancer types, as the authors noted.

There’s still a lot more to this cell death pathway that I don’t know, but from what I have read and learned since last posting, there’s a really cool intersection between the lipid biosynthetic pathways (i.e. ACSL4, enzymes involved in phospholipid synthesis, such as LPCAT–which I didn’t get to talk about), lipid signaling pathways (i.e. 15-LOX and lipid mediators), redox homeostasis (i.e. GPX4 and glutathione), and the ultimate result of ferroptosis. All of which may have impacts in treating diseases where these lipids may play a role, such as cancers. There’s also still a lot of questions that play in my mind too. Since the ferroptotic signals seem to be hydroperoxy-PE species, how do they carry the signal forward to the next actor in the pathway? And what exactly happens next? Which genes are turned on or off? Are there other signaling molecules involved, such as other lipid mediators? Are other lipid biosynthetic pathways involved? (E.g. another interesting article that came out recently found lipid mediators, prostaglandins, could be formed from triacylglycerols during an immune response–this was reminiscent to me on the oxidation of fatty acids on PE in ferroptosis.) As I mentioned in my previous post, oxylipids seem like such an understudied field. And I can imagine why. If the oxygenation is non-enzymatic, then it can be difficult to determine just what the molecular structure is from mass spectrometry alone. Not impossible, but it would take a bit more time and a deeper look into the data (it’s quite literally the equivalent of putting together a puzzle at the molecular scale with mass spectrometry, which is why I enjoy it so much). Enzymatic oxygentation might be a little more predictable in molecular structure, such as 15-LOX that will add a hydroxyl group to the 15^th position of a fatty acid. But even there, since these lipids are more signaling lipids, and therefore low abundant, there’s the difficulty of even “seeing” these lipids as they could be drown out by other more abundant non-oxylipids.

Anyways, I feel like I’m rambling now, so I’ll leave it here. I’ll probably set ferroptosis off to the side for now and look into another lipid class or cell process for my next post. Or I may possibly detail some helpful Python & R scripts that I use when doing lipidomics and mass spectrometry work. Till then.