This Nature study identifies APOE as an interaction partner of human TTYH2, maps their endosomal colocalization and binding site by cryo-EM, and shows that TTYH2 accelerates lipid transfer from APOE-containing lipoproteins to membranes in vitro.
0:00Welcome to Base by Base, the papercast that brings genomics to you, wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. Right now, literally as you're listening to this, Your brain cells are, uh, they are basically screaming for fat.
0:16They absolutely are. It's, you know, your brain is mostly water, but it requires 1000000s of these greasy lipid packages to be delivered every single second just to keep your neurons firing. Right. Just keep your memories intact and everything.
0:29But, I mean, this creates a massive logistical nightmare. Exactly. Because lipids are hydrophobic. They hate water. Yeah, so how do you drive 1000000s of these grease filled delivery trucks through a water-based cellular city without, you know, causing a massive biological oil spill?
0:46Well, we know your body uses these specialized protein trucks to shuttle the fats around safely. But here is the genuine mystery that is just baffled scientists for years. What actually happens when those lipid delivery trucks finally get inside the destination cell?
1:00Right, because how does the cargo actually get unloaded without just completely clogging up the delicate microscopic machinery inside? Okay, let's unpack this because the mechanism taking place inside your cells has been a complete black box until right now.
1:15It truly has. I mean, it's been a missing chapter in our fundamental understanding of human biology. We are talking about a process of life that happens constantly. constantly, yeah. Right? Keeping your nervous system alive.
1:26And yet, the precise mechanics of how these fats are physically extracted from their transport vehicles and integrated into the cells architecture have completely eluded us. We knew the trucks arrived, but we just uh, we couldn't see the unloading dock.
1:41Today we celebrate the work of Anastasia Sukalskaya, Raymond Dutzler, and their colleagues at the University of Zurich and the University of Applied Sciences, Upper Austria, who have advanced our understanding of cellular lipid transport.
1:52We are diving deep into their brilliant 2025 publication in the journal Nature. And I got to say, it reads like a molecular detective story. It really does. And to understand the sheer scale of the problem this team solved, we 1st need a look at the primary delivery truck in question.
2:08It is a highly famous protein called APOE or Apolipo protein E. Right, APOE. Yeah, an APOE is ubiquitous. It operates all over your body, but its role in the brain is absolutely critical. In the brain, it's the main vehicle responsible for shuttling cholesterol and phosphol lipids between your astrosytes, which are the support cells of the brain and your neurons.
2:29And APOE is a name that you might actually recognize if you follow medical news at all, right? Like it has massive clinical relevance. Precisely. If you were familiar with the genetics of Alzheimer's disease, you have likely heard of the APOE 4 subtype.
2:43Oh, right. The APOE 4 variant. Yes. Carrying the gene for the APOE 4 variant is one of the most significant genetic predisposition factors for developing Alzheimer's. So understanding exactly how APOE functions, and crucially, where its delivery mechanism might break down, is just of paramount importance to human health.
3:01Yeah, that makes total sense. And so to set the stage for you, we actually have a pretty good map of how APE navigates up to the cell, right? It drives up to the cell membrane and specialized receptors on the surface, like the low density lipoprotein receptor, they grab onto it.
3:16Exactly. And then the cell essentially swallows the whole truck. It wraps it in a little internal watery bubble called an endosome. Right, endocytosis. We understand that part. But once that endosomal bubble pinches off and moves deep into the cell, that's where the lights go out.
3:33They do. Like the intracellular steps to actually get the lipids out of the APOE truck and into the cell membrane, total mystery. Which brings us to a really fascinating, and honestly, somewhat misunderstood, family of proteins known as the Tweety homologues, or TTYHs.
3:50Tweety homologues. I love that name. It's a great name. And their history in the scientific literature is quite colorful. When researchers 1st discover these proteins embedded in cell membranes. They were actually misclassified.
4:03Wait, really? What did they think they were? Well, early models suggested they were anion channels. Essentially, tiny pores in the membrane that open and close to let negatively charge ions pass through.
4:14Why did they think that was just a case of mistaken identity? Basically, yeah. It was a logical guess at the time. I mean, membrane spanning proteins often function as channels, and early rather confusing experimental data seem to show some electrical current associated with them.
4:29Oh I see. But his experimental techniques improved, that functional role was completely refuted. They definitely were not ion channels. Okay, so they were back to square one. Right. And the mystery deepened when structural biologists finally got a closer look at the architecture of these TTYH proteins.
4:46They noticed something highly unusual. These proteins possess this incredibly wide, deep hydrophobic cavity. So essentially a giant, greasy pocket. Exactly. A giant greasy pocket. And this pocket extends from the cellular membrane right out into the watery lumin of the endosome.
5:04Just a giant, greasy pocket, sitting there waiting for some grease. You got it. And this structural feature led to a bold new hypothesis. Perhaps these TTYH proteins act as the mysterious machinery that extracts lipids from soluble carrier proteins.
5:20So maybe they are the unloaders. Yes, but this was entirely theoretical. It was a compelling idea. But no one had ever identified what, if anything, these TTYH proteins actually interacted with in a living cell.
5:32They had the supposed machinery, but no known partner. So the researchers have this primary suspect, TTYH, specifically the human variant known as TTYH2. But here is where I really have to push back on how you even begin to study something like this.
5:47It's a challenge for sure. Because you've got a hydrophobic protein embedded in a greasy cellular membrane, constantly shifting and moving around in the dark. How on earth do you isolate it and get a clear enough picture, figure out who it is talking to in the middle of a busy, chaotic cell?
6:03It is an incredibly difficult biochemical hurdle. I mean, you can't just live through a standard microscope. To solve this, the research has utilized an ingenious tool called sea bodies. Sea bodies, like synthetic nanobodies.
6:14Exactly. Synthetic nanobodies. You can think of them as highly engineered, miniature antibodies. Specifically, they develop 2 say bodies. called SB1 and SB2. Okay. And these side bodies are custom designed to bind specifically and tightly to the TTYH2 protein.
6:32They act like microscopic molecular clamps, grabbing onto the wriggling protein and locking it into one fixed conformation so it can actually be studied. Okay, so you clamp the squirming protein to hold it still, but how does that tell you who its partner is?
6:47Well, they use that 1st side body, SB1, almost like a molecular fishing lure. It's a fishing lure. Yeah. They immobilize this sci-buddy clamp on a microscopic resin bead. Then they took human kidney cells, specifically HEK 290 cells, which are a very standard reliable workhorse cell line used in biology labs worldwide, and they broke them open to create a complex cellular soup called a liceate.
7:09Right, just a big biochemical soup. Exactly. They poured this cellular soup over the beads. The somebody reached into that chaotic mixture and clamped onto the native TTYH2 proteins, pulling them out. Okay, I see where this is going.
7:21Yeah, here's the brilliant part of a pull down assay. If TTYH2 happens to be holding hands with another protein at that exact moment, that partner gets yanked out of the soup right along with it. Ah, so they catch the partner along for the ride.
7:35And then they analyze the catch using mass spectrometry. Exactly. And for those unfamiliar, mass spectrometry is basically a high-tech waste station that identifies molecules based on their exact mass and electrical charge.
7:47So they pull out TTYH2, run the catch through the mass spec, and what did they find attached to it? APOE. The delivery truck itself. That was the crucial 1st piece of physical evidence linking the two.
8:00They found the truck parked at the dock. But, you know, in biology, finding 2 proteins in the same net doesn't necessarily prove they are functionally interacting. They can just be bumping into each other randomly.
8:11The researchers needed to prove the physical mechanics of the interaction. Are they actually gripping each other? Right, because proximity isn't proof of an actual handshake. Exactly. So to answer that, they used a technique called single molecule force spectroscopy or SMFS.
8:24Walk me through that because the physics of this just blow my mind. They are literally measuring the physical tug of war between 2 single molecules, right? It is a truly remarkable method. So imagine an atomic force microscope.
8:37It features a microscopic cantilever. Think of it like a tiny, incredibly sensitive diving board. Or like a microscopic record player needle. Yes, exactly like that. At the end of this diving board is a probe with a tip so unbelievably sharp it can interact with individual molecules.
8:54The researchers covalently attached a single TTYH2 molecule right to the end of this tip. Okay. Then they firmly glued APUE molecules onto a glass floor beneath it. So you've got TTYH2 on a microscopic needle pointing down, and APOE glued to the floor facing up.
9:12Correct. They lower the diving board until the 2 proteins meet and bind together. Then they slowly retract the board, pulling it upward. And because they're stuck together. Right, because the proteins are holding onto each other, the tiny diving board actually bends.
9:24By measuring the exact angle of that bend before the proteins finally snap apart, they calculated the mechanical force of the bond. That is insane. What do they find? They found an off rate constant of about one per second.
9:36Okay, let's translate that metric for a second. An off rate constant of one per second. What does that actually look like inside the cell? It means the bong is highly specific, but transient? It's not a permanent super glue.
9:49When TTYH2 and APOE meet. They grip each other for about a second. A whole second. Yeah, that is just long enough for the biochemical transaction to occur, but dynamic enough that the empty truck can then release and drive away to make room for the next one.
10:04It is a perfectly tuned biological docking mechanism. That is incredible. Okay, so the mass spec proved they are in the same room. The 4 spectroscopy approved they actually grip each other. But I know they also wanted to take a literal picture of this happening.
10:18They did. And to do that, they turned to cryo-EM or cryogenic electron microscopy? Yes. Cryo-EM involves flash freezing the protein complexes so rapidly that damaging ice crystals don't even have time to form.
10:32It traps the molecules perfectly in their natural state. Then, you bombard them with electrons to generate high resolution 3D structures. But, as you hinted at earlier, getting a picture of this specific interaction was a nightmare.
10:46Right, because APOE is apparently a very floppy, flexible truck. In the paper, they call it confirmational heterogeneity. I imagine it like trying to take a crisp panoramic photograph of a toddler who absolutely refuses to stop squirming.
11:00That is exactly the problem. That flexibility is a defining hallmark of abolipa proteins. They actually have to be floppy so they can change shape to wrap around different sizes of lipid cargo. Oh, that makes sense.
11:11But that constant skirming makes structural biology exceptionally challenging. Because cryo EM relies on averaging 1000s of images of the protein in the exact same pose. A squirming protein just blurs the final image.
11:24But despite the squirming, they manage to get a structure of TTYH2 alone at a 2.7 Angstrom resolution. He did, which is phenomenal. Yeah, to put 2.7 angstroms into perspective for you. And Angstrom is 110 billionth of a meter.
11:39Zooming into 2.7 Angstroms means they aren't just seeing the general blob of the protein. They are seeing the individual atomic branches of the amino acids making up the structure. And at that astonishing level of detail.
11:52The map showed exactly how this unloading mechanism functions. TTYH2 has an extracellular domain, a sort of molecular arm that reaches out into the water illumin of the endosome. Okay. This is the part that physically latches onto the APOE truck.
12:08But the real revelation was the lipid pathway itself. Right. So if APOE is the delivery truck. I initially thought of TTYH2 as a forklift. But looking at how the cargo actually moves in these structures, it's not really lifting anything, is it?
12:21No, your earlier analogy of a forklift definitely needs an upgrade. The structure revealed what the researchers describe as a continuous lipid belt. Yeah, TTYH2, physically, grabs lipids and guides them along a pathway.
12:33If you were to look at a normal cell membrane. The lipids sit vertically, standing side by side like people in a crowded elevator. Right, forming that barrier. Exactly. But the cryo EM structure shows that as the lipids interact with TTYH2, the protein forces them to tilt.
12:49It acts more like a luggage carousel on a slant. Oh, wow. It grabs the upright lipids and gradually forces them into a horizontal orientation, allowing them to slide smoothly off the APOE truck and straight into that large, greasy cavity we talked about earlier.
13:04It's literally a molecular slide for fats. Exactly. But wait, I'm getting stuck on how they prove the fast actually travel down the slide. To test the performance, the researchers used and in vitro for 8 assay.
13:15Freck stands for 1ster resonance energy transfer. Correct? So they put fluorescently glowing lipids into the APOE truck. But if you just dump glowing fats into a cell, doesn't the whole thing just light up?
13:26How does that prove the lipid actually moved into the TYH2 cavity? That is a great question, and it really highlights the cleverness of this asset. They didn't just watch it glow. They watch it go dark.
13:36Go dark. Yeah. They created artificial endosomes called liposomes. And crucially, they built the walls of these liposomes using a specific lipid called DPPC. Why DPPC? What special about that? Because regular cell membranes are highly fluid.
13:51They are like a wobbly waterbed. Lipids naturally jump in and out all the time, which creates massive background noise. Right, making it hard to see a specific transfer. Exactly. But DPPC builds a very stiff, quiet membrane.
14:04It acts like a solid concrete floor, which reduces the background noise of random lipid jumping to almost zero. And inside these quiet TTYH2 liposomes, they placed a quenching molecule. Ah, I see. So they load the glowing fats onto the APOE truck, the truck docks with T2YH2.
14:22And if TTYH2 successfully slides that fat into the liposome, the quenching molecule instantly turns the light off. Exactly. The rate at which the lights went out told them exactly how fast the unloader was working, and the results were stark.
14:36How fast are we talking? Well, the presence of TTYH 2 didn't just allow livid transfer to happen. It actively accelerated the transfer rate by a massive 14 fold compared to the background rate. 14 full. Yeah, it doesn't just sit there passively.
14:48It is highly efficient, active catalyst pulling the fats inside. And we should definitely clarify what it is not doing. right? Because there was a competing theory out there, that TTYH might be a scrambleize.
14:59And for context, a scramblase is a type of protein that violently flips lipids from one side of a membrane wall all the way to the other. Right, and the researchers tested for that exact activity, and the data firmly proved TTYH2 is not a scrambloss.
15:14So no flipping. No flipping. It does not flip lipids across the membrane leaflets. It is strictly an unloader, pulling lipids from the APOE carrier and gently inserting them into the outer layer of the membrane.
15:25such a crucial distinction. It is. They also tested its sibling protein, TTYH3. Despite being in the exact same genetic family, TTYH3 did not bind to APOE, did not accelerate the transfer of the fats. Oh fascinating.
15:41It really highlights the exquisite evolutionary specificity of TTYH2. It evolved for this exact job and only this job. Before we wrap up the structural stuff, we also have to mention that they visualize this whole process happening in the living cells, right?
15:53Yes. Using convocal fluorescence microscopy. They tag TTYH2 with a green glowing marker, an APOE with a red one. Like little biological traffic light. Exactly. And when they looked at living sales under the microscope, specifically looking at the endosomes, they saw green and red, literally glowing together, perfectly colloquialized, right inside the compartment.
16:16That's the ultimate proof. It really is. It confirmed that everything they measured in the test tubes and with the atomic force microscope wasn't just some artificial laboratory trick. This dynamic docking is happening exactly where it is supposed to happen inside the living cell.
16:30So if we step back for a second. If we connect this to the bigger picture, Why should we care about this highly specific unlearning process? Because while this lipid delivery process is happening in cells all across your body, it is particularly critical in your brain.
16:46The brain is uniquely, hungrily dependent on a constant, highly regulated flow of cholesterol and phosphil lipids to build mylin sheets and maintain neural networks. An APOE is the truck. Yes. And if TTYH2 is the primary unloader for APOE in these cells.
17:02Any bottleneck, any genetic mutation, or any mechanical malfunction in this interaction, could cascade into massive cellular starvation. Oh wow. This ties directly back to why variations in APOE are so heavily implicated in neurodegenerative diseases like Alzheimer's.
17:18If the delivery trucks arrive, but the molecular slide is broken. The lipids just pile up in the end of sun. A literal traffic jam. Exactly. The cell starves for its necessary building blocks, traffic jams form, and the entire neural system becomes compromised.
17:31It is deeply profound to think that a disease as devastating and complex as Alzheimer's could have its roots in a simple mechanical failure at a microscopic cellular learning dock. It really is Now, looking at where the science goes next, you mentioned TTYH3 didn't bind to APOE.
17:48What are the other siblings doing? That is the next great mystery. If TTYH2 binds APOE, what are TTYH1 and TTYH3 doing? What specific lipid carriers do they interact with? Right, they must have their own specific trucks.
18:00Exactly. Furthermore, while this study brilliantly outlines the mechanics in isolated cells and highly controlled biochemical assays, the crucial next step is translating this into live, complex brain tissue models.
18:11We need to observe this TTYH2 APOE dynamic within the fully functioning architecture of the central nervous system to see how it reacts under physiological stress. To sum it all up, this research solves a massive biological mystery by revealing that TTYH2 acts as a highly specialized, active, cellular unloader.
18:31It interacts directly with the APOE delivery truck inside the sales endosomes, gripping it to extract lipids down a molecular slide and insert them safely into the cellular membrane. By doing so, it finally illuminates the long missing, critical, intracellular step of how our cells actually receive the fats they need to survive.
18:49What does this mean for our future ability to treat nodegenerative diseases? If we can target the cellular loading dock itself, could we one day clear the lipid traffic jams that lead to Alzheimer's? This episode was based on an open access article under the CCBY 4.0 license.
19:04You can find a direct link to the paper and the license in our episode description. If you enjoyed this, follow or subscribe in your podcast app and leave a 5 star rating. If you'd like to support our work, use the donation link in the description.
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