Acetylation and aging

For a very long time, aging was considered to be largely the result of wear and tear accumulating over a lifetime — be it free radicals, accumulated mutations, or even the rate of living hypothesis, all of them assumed unrepaired damage accumulate over time, eventually culminating in irreversible functional compromise. Until we found the genes that can extend lifetime — the sirtuins. So what do sirtuins do? Lo and behold — deacetylation. …Are you intrigued yet or are you scratching your head? Well, I certainly was disappointed at first. Throughout my molecular biology and physiology modules, never was acetylation even discussed as something important to the regulation of anything. But that was just the ignorance of textbooks. So what do acetylations do? Here’s the story.

The road taken by carbohydrates and lipids to become cellular energy converges at a molecule called acetyl-CoA. It is a two-carbon unit that forms in the mitochondrial matrix. Acetyl-CoA can then finally join the TCA cycle to be oxidized completely to carbon dioxide that we breathe out. Acetyl-CoA is essentially an activated form of an acetyl group, with a "high energy" thioester bond (high energy means the free energy released by the bond hydrolysis is high, because of the poor orbital overlap between oxygen and sulfur due to their size differences). Otherwise, the carboxylic acid acetate is rather chemically inert — this is why your metabolism likes to skip reactive aldehydes quickly to make stable carboxylic acids (many metabolites are carboxylic acids, but very few are aldehydes. Whatever little aldehydes that form e.g. glyoxal, formaldehyde, acetaldehyde make you sick if they accumulate e.g. hangover). This is the same strategy used by organic chemists to force reactions — one must use the activated form of acetate, such as acetic anhydride if one wants to add those two carbons onto something else. The anhydride group is too reactive for biochemistry, so a phosphate group or coenzyme A is usually used as a good leaving group. Acetyl-phosphate does exist as a metabolite in bacteria such as E. coli, but it is still too reactive for the eukaryotes so glycolysis is rewired to avoid it (at the expense of ATP yield). Nevertheless, when accumulated in high concentration, even acetyl-CoA can start reacting with nucleophiles — in biochemistry, that is usually the lone pair of nitrogen, so acetylation happens on nitrogen-containing lysine residues.

When I was first taking biochemistry, I remember the teacher told us that non-regulatable non-enzymatic reactions are usually not favorable in the body and very few of them take place. He said he only knows two — one is the spontaneous decarboxylation of beta-keto acids such as the ketone body beta-hydroxyglutarate (this is why patients with diabetic ketoacidosis breathe out the sweet smell of acetone), and the other is non-enzymatic glycation due to hyperglycemia. Well, there’s another — acetylation in the mitochondria. The mitochondrial matrix pumps protons to the intermembranous space to create a proton gradient that drives life itself, and as a result, the mitochondrial matrix is comparatively more alkaline than the cytoplasm. This keeps the lysine residues deprotonated and the lone pair free to react with acetyl-CoA. So when acetyl-CoA concentration spikes inside the matrix, spontaneous acetylation can take place. Indeed, an estimated 35% of mitochondrial proteins are acetylated. Acetyl-CoA in the mitochondria can also be transported into the cytoplasm via the citrate shuttle (one of the most important shuttles these days, regrettably doesn’t get much attention in textbooks). In the cytoplasm, however, acetylation reactions are much more regulated, as the concentration never gets that high and pH lower than in the matrix. There are specific acetylation enzymes that mediate protein acetylation. Cytoplasmic proteins and histones are acetylated in this manner.

When does the acetyl-CoA level get so high? When there are excessive nutrients. Fatty acid oxidation produces particularly a lot of acetyl-CoA, so if the cell is put under a high fat supply, protein acetylation increases. This also means that proteins being acetylated carry information about nutrient availability — and there are very few things as important to a cell as the nutrient availability of the environment! So life evolved to use acetylation as a sort of signaling, that the current environment is rich in energy substrates, and the time for investment is now. “Grow, divide, make kids!” When mitochondrial proteins are acetylated, respiration is suppressed, as excess ATP would cap the flux of biosynthetic reactions. When histones are acetylated, genes that promote growth and proliferation are activated. But this state sacrifices the longevity of the cell. After all, if the time is now, who cares about maintaining cellular machinery for later? As long as the kids made during the time of abundance have a better chance of survival, the genes survive with them, so the old vehicle for genes doesn’t need to stay around anymore. By now you might be guessing — if acetylation makes us age faster, what about deacetylation, will it make aging slower? Yes. Enter, sirtuins.

Acetyl groups themselves are terrible leaving groups, so deacetylation requires a special group of enzymes, which are sirtuins. When you’re hungry a lot, the energy-expanding “grow, divide, make kids!” message of acetylation is no longer relevant, so cells must reverse it. Deacetylated state, therefore, carries the opposite message — “maintain the status quo, lay dormant, endure, wait for a better time” — and this message activates longevity. We get old because we know the nutrients around us were sufficient to allow the survival of our offspring, so we lose the meaning to keep living. We stay young if the environment is barren.

How do sirtuins know the time is bad and caloric intake is low? Sirtuins use NAD+ as their co-substrate — this is impressive in two ways, first because NAD+ is actually chemically capable of helping in deacetylation reaction, as it has an electrophilic spot for with a good leaving group (and ingeniously, the leaving group is only good if it’s charged, i.e. in its oxidized form, making NADH incapable of doing the same chemistry), and second because cellular NAD+ level is tightly correlated with nutrient availability. Why does carbon dioxide have no energetic value to a cell? Because carbon dioxide is fully oxidized, all four bonds that can be formed by a carbon atom all bonded to oxygen. Life is a redox reaction, and the business of living rests entirely on the journey of “electrons looking for a place to rest” (Sczent-Györgi). Thus what we call “food” are not exactly energetic because they contain carbons, but because those carbons are in reduced states, still capable of going down the redox hill to release energy in the process. Before the redox energy in food is eventually converted to proton gradient and ATP, it rests briefly on “reduced equivalents”, or molecules that can be temporarily reduced to transfer the redox energy to the proton gradient. So the more food we eat, the more “reduced equivalents” become reduced. The hungrier we get, the more “reduced equivalents” get oxidized. NAD+/NADH is the main reduced equivalent in a cell, and NAD+ is the oxidized state, NADH is the reduced state — if you have too much NAD+, it means the nutrient is insufficient. Sirtuins get lots of NAD+ as their co-substrate, and it turns on to save us from aging too soon.

A question comes to mind at this point — if increasing NAD+ increases lifespan and decreasing NAD+ decreases lifespan, then perhaps normal aging is related to decreasing NAD+ levels? That is indeed correct. But why? NAD+ level decreases because something called CD38 increases over age. CD38 is found on immune cell surfaces and is related to inflammation — aging is actually a slow constant state of inflammation! But at this point, we are leaving acetylation as a topic, so I will leave the rest of the story for another time.