This study identifies crotonylation as a posttranslational modification of c-Myc that reduces its transcriptional and oncogenic activity. Key lysines K289 and K298 are crotonylated; loss of crotonylation (including a cancer-derived K298N mutant) enhances Skp2 binding and tumorigenesis.
0:00Welcome to Base by Base, the paper cast that brings genomics to you wherever you are. Thanks for listening, and don't forget to follow and rate us in your podcast app. Imagine you have a factory manager inside your cells.
0:12Okay. And this manager is just incredibly powerful. I mean, they control the expression of like 15% of all your genes. is a massive amount for one entity. Right. They're in charge of television, metabolism, building cellular machinery.
0:26Basically everything you need to keep the assembly line moving. But um, there's a problem. usually is. Yeah. This manager is overzealous. Like they completely refuse to stop the line. In fact, this single hyperactive factory manager is responsible for driving 70% of all human cancers.
0:43Wow. 70%. I mean, it is just a terrifying level of control for one single molecule to possess. Exactly. And for decades, scientists have been trying to figure out how to, you know, fire this manager, or at least slow them down.
0:56But what if the physical break for this deadly cancer protein isn't, um, isn't some synthetic drug we have to invent in a lab? What if that break is actually controlled by a short chain fatty acid? And not just any fatty acid, but one that can be produced right inside your own gut.
1:12Like, by the bacteria fermenting the dietary fiber you ate for breakfast? I mean, that completely reframes how we think about the relationship between our diet, our microbiome, and really the most fundamental mechanisms of cellular growth.
1:26It really does. So the mission of this deep dive is to explore this newly discovered cellular modification. We are going to look at exactly how it acts as a physical break on cancer growth. The, um, the really elegant science used to uncover it and the devastating consequences of what happens when that break is removed.
1:45And to do that. Today we celebrate the work of Walloo, Nicholas J. Wall Village, and their incredible team at the Tulane University School of Medicine and Tulane Cancer Center, who have really advanced our understanding of oncogene regulation.
1:57Yeah, their work is fantastic. It is. Their research paper, which was published in the proceedings of the National Academy of Sciences in June of 2026 provides the roadmap for this entire discussion. Okay, let's unpack this.
2:09The factory manager we're talking about is a protein called C Mike. Right, C Mike. It's a master transcription factor. Its entire job is basically to bind to your DNA, and read the instructions to make other proteins.
2:23And when it is overexpressed, it drives incredibly aggressive cancers like lung, breast, colon, leukemia. Yeah, the big one. Exactly. And historically, C Mike has been considered, quote unquote, undruggable.
2:36But why is that? I mean, we have drugs for so many other proteins, right? Yeah, we do, but that undrug ability comes down to its physical structure. See, many proteins fold into very specific, um, rigid three-dimensional shapes.
2:50And because they're rigid, they have these little crevices and pockets on their surface. So you can design a small chemical drug to fit perfectly into one of those pockets, like a key sliding into a lock.
3:01Exactly. Like a key to a lock to just jam up the machinery. But seeing my car is what we call an intrinsically disordered protein. So it doesn't have a rigid shape. It's more like, I don't know, like a piece of wet string just constantly wiggling around.
3:13Precisely. It's flexible. It's chaotic. And because it's constantly shifting. It doesn't have those stable pockets for a drug to bind to. I mean, you can't design a key if the lock is constantly changing shape.
3:25That makes total sense. So if we can't drug the C mic engine directly from the outside, We're sort of forced to understand how the cell naturally regulates its speed from the inside. Right. Because if the cell is constantly producing this chaotic, you know, wet string that wants to force the cell to divide.
3:41How does it avoid a total meltdown? Yeah, there has to be an internal emergency break somewhere. There is. And it involves something called post-translational modifications or PTMs. Okay, PTMs. Let's define that.
3:54Think of a PTM as a chemical tag that gets added to a protein after it's already been built. The cell attaches these tiny chemical groups, like a phosphate group, or maybe ubiquitant tag 2 specific amino acids on the protein chain.
4:08So they act like tiny switches. Exactly. They can change the protein shape or tell it's a move to a different part of the cell or crucially market for destruction in the cellular recycling center. So we have these chemical tags trying to control the C mic engine.
4:22And one of the main regulators doing the tagging is this enzyme called SKP2. Right. SKP 2 is a ubiquit in Legus. Right. And its job is to attach a ubiquitant tag to see Mike, which, as you said, is basically the universal cellular signal for destroy this protein.
4:37But looking at the literature, SKK2 presents us with this wild biological paradox. Oh, it truly is one of the most fascinating mechanisms in cellular biology. So SK2 binds to see Mike and tags it for destruction.
4:52But here's the crazy part. That very act of binding and tagging, that physical interaction is actually required to stimulate C Mike's cancer driving activity in the 1st place. Wait, let me make sure I'm picturing this right.
5:04The very thing that destroys CMike is also the thing that turns it on. So SKP 2 is essentially a gas pedal that burns out the engine. Like it ramps seem like up to maximum speed, forces it to read the genes that cause cell division, and then immediately sends it to the Shredder.
5:17Exactly. It guarantees that CMI can only act in very short, powerful bursts. You get a spike of cell division signals, and then the signal is destroyed before it gets out of control. Oh, I see. actually pretty brilliant.
5:29It is, normally. But in many cancers, this balance is lost. The shredder can't keep up, and SCOP2 actually acts as an enco protein itself. It's just constantly turning the key in the C mic ignition. Which means the cell needs a secondary layer of control to stop SKP 2 from constantly hitting the gas, and that leads to the core discovery from the Tulane team.
5:50They found a totally new post-translational modification acting on CMI called crotonilation. Yeah, quote annihilation. It's a specific form of vacillation. Basically, it involves attaching a molecule called crotonade, which is a short chain fatty acid onto the protein.
6:05Okay, but we knew about annihilation before this, right? We did, but historically, we knew it happened on his stones. Those are the spool like protein that your DNA wraps around to stay organized. Finding this fatty acid tag on a non-histone protein, especially a chaotic master oncogene like CMIT, there's a massive shift in our understanding.
6:24Okay, but crotenate is tiny. I mean, it's just a few carbon atoms. How do researchers physically find a tiny fat molecule attached to a massive protein inside a living messy cell. Right. a needle in a haystack.
6:37But the methodology here is incredibly elegant. The researcher started with human cancer cells. Specifically, HCT 116 colon cancer cells and HEK 293 embryonic kidney cells. Okay. They exposed these cells overnight to crotonate, and actually see if the modification was happening.
6:58They used pan crotonnihilation antibodies. Antibodies, like what our immune system makes. Exactly, but custom designed. They act like highly specific homing beacons. They're engineered to bind only to proteins that have a Krotonite tag attached to them.
7:11Oh, okay. So they use these antibodies to basically pull out the tagged proteins, and then they run them on a gel, and they can physically see a band indicating that, yes, CM Nules has crotonate attached to it.
7:22Yeah, exactly. But wait, C Mike is a chain of over 400 amino acids. Just knowing the tag is somewhere on the string, doesn't really help us understand how it works. How do they pinpoint the exact location?
7:32For that, they utilize high resolution mass pick traometry, or HRMS. Okay, I've heard of that. How does it work here? Mass spectrometry, essentially acts as a highly advanced molecular scale. They take the Crot annihilated C mic protein and use enzymes to chop it up into tiny predictable fragments.
7:49Like molecular scissors. Right. Then they shoot those fragments through the mass spectrometer, which weighs them with extreme precision. Oh, I get it, because they know the exact mass of a normal C mic fragment, and they know the exact mass of a krautinate tag, so they're just looking for the fragment that weighs slightly more than it should.
8:07Precisely. The HRMS reads the sequence of those heavy fragments and pinpoints exactly which amino acids are carrying that extra weight. And they found that crotonylation only happens on very specific amino acids called lycines.
8:22Lycines. Okay. But, you know, correlation isn't causation. Just knowing the fatty acid attaches to a lysine doesn't prove it acts as a break on cancer. No, it doesn't. You have to physically remove the brake to see if the cell crashes, right?
8:35That is exactly where they employed a classic molecular biology technique called PCR mutogenesis. Okay what is that? They went into the genetic code for C Mike. And literally swapped out the instructions for those specific lacine amino acids, changing them to a different amino acid called arginine.
8:54Why Arginin? Why not just, I don't know, delete that section of the protein entirely? Because if you delete a section, you risk completely destroying the overall three-dimensional behavior, the protein.
9:05The string might just collapse, which would ruin the experiment. Oh, right. break the whole thing. Exactly. Arginine is the perfect molecular decoy. It's very similar to lycine and its physical size and its positive electrical charge.
9:17So it behaves the same way in water. Right. Swapping them doesn't fundamentally change how the wet string behaves. However, Argentine lacks the specific chemical hook that the crot nay tag attaches to.
9:29Ah. So by swapping lycine for arginine, you create a crotonnihilation deficient mutant. Exactly. The cell physically cannot attach the crotonay brake to those spots anymore. So the researchers create these unbreakable mutants.
9:42And I read they made an 8 R mutant, where they swapped out 8 different lycines for Arginines, and a 2 R mutant, where they only swapped out 2 specific lycines. Yes. So what actually happens when you put these mutant proteins back into living cancer cells?
9:59To measure that, they used incusite proliferation assays. and colony formation assays. Let's break those down. Sure. And Incusite system is essentially an incubator with a built-in automated microscope.
10:10Oh, that's cool. So it's taking pictures constantly. Right. It takes pictures of the living cells every few hours over several days. This allows researchers to calculate exactly how fast the cancer cells are dividing and multiplying.
10:21And the colony formation essay. that looks at the long term. Can single cancer cells survive and grow into large visible colonies over a couple of weeks? Both of these are direct measures of oncogenic power.
10:33And the results were just explosive. I mean, the mass spectrometry originally found 10 different crotonihilation sites on C mite. But through this careful mutation process, the researchers narrowed it down to 2 critical linchpins, Licing 289 and License 298.
10:48Right. When just those 2 specific sites, K289 and K298 cannot be crotoniliated, the cancer cells gain a massive proliferative advantage. Wow, just 2 tiny spots. Yeah. And it didn't matter if they used the 8R mutant or the highly targeted 2R mutant.
11:04The effect was exactly the same. The cells divided significantly faster in the Incusite. They form significantly more colonies. Did they check which genes were being activated? They did. When they extracted the RNA to see what Gene C Mike was turning on.
11:16They found specific genes responsible for cell division and survival were completely hyperactivated. Okay, so removing the brake at just those 2 specific spots puts the cancer engine into overdrive. But I want to go back to our analogies.
11:28If CMike is a chaotic wet string. How does attaching a tiny fat molecule to it actually stop it from driving cancer? Like what is the physical mechanism there? To answer that, the researchers turn to alpha fold, which is an advanced artificial intelligence program.
11:42Wait, the protein folding AI. Exactly. You feed alpha fold in amio acid sequence, and it predicts how those electrical charges and repulsions will cause the protein to crumple and fold in three-dimensional space.
11:53And what did Alpha Fold predict when it looked at C Mike with these crotonit fats attached to it? It predicted a radical structural shift. The crotonellated C mic becomes much more compact. It essentially loses a lot of its intrinsically disordered nature and folds up tightly.
12:08Okay, let me guess. If the string frumples up into a tight ball, it physically hides the binding site for SKP2. The Shredder can't get a grip to turn the ignition. That is exactly the mechanism they elucidated.
12:21Crot annihilation acts as a physical barrier. It changes the confirmation of C Mike, just enough to block SKP 2 from binding. Wow. And remember this KP2 paradox we discussed earlier. SKP2 needs to bind to CMIGA to license it to activate that massive burst of transcriptional activity.
12:38If quotinate is attached, as KP2 can't bind, the gas pedal can't be pressed, the cancer genes stay quiet. But if you look at the uncrotonated C mike like are 2R mutant where the brake is removed and the string stays loose.
12:51Does it bind to SKP too? It binds incredibly strongly. And the researchers prove this through immuno precipitation essays. Which is where you pull one protein out of a cellular soup and see what else is stuck to it, right?
13:04Exactly. And the uncoordinated C mic, pulled down significantly more SKP2. That strong binding ramps up the cancer activity to dangerous levels, while, paradoxically, decreasing the overall lifespan of the C-Mike protein itself.
13:19Because SKP 2 is still tagging it for the Shredder. Right. The engine just does an incredible amount of damage before it gets shredded. Man, this is where the paper moves from, you know, fascinating molecular biology to something with profound human implications.
13:33Because you might be listening to this thinking, okay, that's what happens when scientists engineer an artificial mutation in a petri dish. Does this matter for real patients? Which is exactly what the researchers asked.
13:44Right. They went looking through genomic databases of actual human cancer tumors. And they found a naturally occurring mutation at this exact spot at Lycine 298. It's called the K298 end mutation. In real human patients, the lycene at position 298 naturally mutated into a different amino acid called asparagene, and they found this specific mutation in primary human lung and breast tumors.
14:09That is wild. The tumor itself figured out how to remove the brake to survive. It's the ultimate chilling validation of the petri dish findings, and to prove just how dangerous this natural human mutation is.
14:20The researchers used an invivo mouse xenographed model. Let's explain what that is for the listener. A xenograph is a classic oncology model. You take mice that have been specially bred to have suppressed immune systems.
14:32Yes, so they won't automatically reject foreign human tissue. Right. And you inject human cancer cells into them to see how the tumor behaves in a living, breathing body with a circulatory system. Exactly.
14:42So they injected these mites with human lung cancer cells. On one flank of the mouse, they injected cells with normal, wild type C might go. On the other flank, they injected cells engineered to carry that natural human K298 and mutation.
14:56Okay, side by side, what happened? After 4 weeks, the difference was staggering. The mice with the K298 and mutant grew significantly larger, heavier tumors compared to the wild type. Did they look at the tissue itself?
15:10They did. When they examined the tumor tissue, the K298 and tumors had drastically higher levels of a protein called Kai 67. And Kai 67 is a key marker for cellular proliferation. It's basically a flag that sells wave only when they are actively dividing.
15:25So more Kai 67 means a much more aggressive tumor. Exactly. They proved definitively that losing this single crotonnihilation site, turbocharges tumor formation in living bodies, a single amino acid change, and the cancer just goes wild.
15:39If we connect this back to you, the listener, the implications are just vast, because we have to ask the most obvious question, where does crotonate actually come from? I mean, our bodies don't just magically summon complex fatty acids out of the void.
15:49No, they don't. Crotonate is a metabolic intermediate, and one of its primary sources is actually our gut microbiota. Wait, really? Our gut bacteria. Yes. Specific strains of bacteria that live in your colon right now, like acid and metococcus fermentans and various clustridia species.
16:07They generate crotonate as a byproduct when they ferment the dietary fiber you eat. Hold on, let's trace this pipeline. Are you saying that I eat fiber for breakfast, the bacteria in my gut ferment it?
16:18They release crotonate, and that molecule somehow travels all the way to my lungs to act as a physical break on one of the deadliest cancer proteins in existence? I mean, that is the biological pipeline the data points toward.
16:31The crotony produced in the gut can passively diffus into your intestinal cells or be transported through the bloodstream. That's incredible. It gets better. Once inside a cell, an enzyme called ACSS 2 grabs the Crodenade and converts it into its active form, Cartonal CoA.
16:46Does our body make it on its own too? It does. It's also produced endogenously by your own cells as a byproduct of mitochondrial metabolism, specifically when your body breaks down fatty acids or amino acids.
16:57Okay, so there are 2 sources. Right. And the overall interacellular concentration of this crotonal coA is the primary driver of how much crat annihilation actually happens on proteins like CMI. This raises a massive, tantalizing question for the future of medicine.
17:13Could we one day alter a patient's diet, or, you know, give them a highly specific probiotic to purposefully increase the population of carotonate producing bacteria in their gut. It's huge possibility.
17:25Or could we design a therapy that stimulates that ACSS2 enzyme you mentioned to pump out more of these tags, effectively forcing the brakes onto semic and starving the tumor. It is an incredibly promising therapeutic avenue, but as scientists, you know, we have to approach it with rigor and acknowledge the limitations of the current study.
17:41Fair enough. What are the limitations? Well, we have to remember that protein regulation is incredibly complex. When the researchers use PCR metogenesis to substitute lycine for arginine, they prove that specific location is critical.
17:54However, Lysine 289 and 298 exist in a region of C Mike that is heavily trafficked by other modifications. Oh, I see. It's like a busy intersection. There's competition for that exact parking spot on the protein.
18:07Exactly. Changing that lacine might accidentally block other PTM tags that normally attach to those exact same spots, like acidillation or sum oilation. Ah, so the explosive cancer growth we observed with the 2R mutant might be partially due to the loss of those other regulatory tags as well, not just crotonate.
18:25Precisely. Future research has to untangle these highly specific overlapping modifications to see exactly how much of the braking power belongs strictly to crotenate. And we also need to find the cellular machinery actually doing the tagging, right?
18:39Yes. We know ACS has 2 provides the raw material, the Cottonal CoA. But we do not yet know the specific enzymes, the quadrant trance races, that physically pick up the tag and attach it to CMI. And what about taking it off?
18:53Right, nor do we know the specific enzymes, the decrotney lases that might remove it. Finding those specific transfrays enzymes is the next great hurdle. Why are those enzymes so important to find? Because enzymes have rigid structures with defined active sites.
19:08They're historically much, much easier to target with small molecule drugs than disordered proteins like CMA. Oh, so if we can find the enzyme that attaches the crotonet brake, we can design a drug to hyperactivate that enzyme.
19:21Exactly. Speaking of drug ability, there's actually a structural silver lining here regarding C Mike itself, we established earlier that C Mike is undruggable because it's a chaotic, wiggling wet string.
19:33But alpha fold predicted that when C Mike is crotonalated, it folds into a more compact, rigid structure. Yes, and that compaction could be a game changer. A compact, rigid structure often creates the stable pockets and crevices required for traditional small molecule drugs to bind.
19:51Wait, really? Yeah. It's entirely possible that by forcing CMIC into its annihilated state, we don't just stop CK2 from binding. We might actually force C Mike into a shape that finally has a keyhole, making the quote unquote undruggable protein vulnerable to traditional pharmacology.
20:06Man, it is just a brilliant intersection of metabolism, structural biology, and oncology. It really is. To distill everything we've covered today. Crotonylation is a newly discovered chemical modification that acts as a vital physical break on C mic, which is one of the most dangerous cancer driving proteins in human biology.
20:25It works by compacting the protein's chaotic structure. Physically blocking its interaction with the coactivator SKP2. But when cancer cells naturally mutate to prevent this modification as seen in real human lung and breast tumors, that brake is permanently removed, an aggressive tumor growth goes into absolute overdrive.
20:42What does this mean for the future of cancer treatment, and could the secret to suppressing our deadliest onca genes be hiding right now in our own microbiome? This episode was based on an open access article under the CCBY 4.0 license.
20:55You 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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