Ricin: nature's ninja

I've been catching up on Breaking Bad over the break, and it has been...wow. Talk about taking a very un-Mythbusters route to making science interesting. Characters in the show make meth, explosive crystals, and the perfect cup of coffee...all using chemistry!

He's like Bill Nye, but more murder-y (Photo by Popculturegeek.com via Flickr)

One wonder of science that keeps popping up in the show is the very potent poison ricin. The main character Walter White purifies the toxin from castor beans, then employs it in a number of dirty deeds that I won't mention. This got me wondering about how ricin works as a toxin, so I hopped on Wikipedia and checked it out. Turns out ricin is AMAZING at what it does, namely killing almost anything.

"Big deal" you say, "there are plenty of things that can kill you."

Yes, but a lot of poisons are like Rambo: they bust into your body, whip out a bazooka, and fire at random, reaping havoc on anything and everything in your body.

Ricin is different. Ricin is a poison that only nature could have designed. It slips past every defense the cell has. It uses the cell's machinery against it. Then it destroys the cell from the inside. Ricin is nature's ninja.

The spaghetti-like face of death (via Wikipedia)

Want the details? Here's my translation of Wikipedia's explanation of how ricin works. Feel free to skip over the techno-jargon:

Ricin is classified as a type 2 ribosome inactivating protein (RIP). Whereas Type 1 RIPs consist of a single enzymatic protein chain, Type 2 RIPs, also known as holotoxins, are heterodimeric glycoproteins. Type 2 RIPs consist of an A chain that is functionally equivalent to a Type 1 RIP, covalently connected by a single disulfide bond to a B chain that is catalytically inactive, but serves to mediate entry of the A-B protein complex into the cytosol. Both Type 1 and Type 2 RIPs are functionally active against ribosomes in vitro, however only Type 2 RIPs display cytoxicity due to the lectin properties of the B chain. In order to display its ribosome inactivating function, the ricin disulfide bond must be reductively cleaved.^[12]

Ricin kills cells by inactivating the cell's ribosomes, the protein-factories of the cells. Since most do most of the moving-and-shaking in the cells of the body, blocking the ribosome is kind of like cutting out the heart of the cell. Everything stops.

All ribosome inactivating protein (RIP, appropriately) have a part that inactivates ribosomes, but some of them (type 1) aren't poisonous. They can even be found in barely, a key ingredient in beer (which can be poisonous, but for unrelated reasons). On the other hand, type 2 RIPs like ricin are very toxic. That's because, in addition to a ribosome-inactivating chain, they've got a lectin (sugar-binding) chain.

Entry into the cytosolThe ability of ricin to enter the cytosol depends on hydrogen bonding interactions between RTB amino acid residues and complex carbohydrates on the surface of eukaryotic cells containing either terminal N-acetyl galactosamine or beta-1,4-linked galactose residues. Additionally, the mannose-type glycans of ricin are able to bind cells that express mannose receptors.^[16]Experimentally, RTB has been shown to bind to the cell surface on the order of 10⁶-10⁸ ricin molecules per cell surface.^[17]

The cell surface is covered in chains of sugars. They help cells ID each other, allow select proteins to bind to the cell, and perform a host of other functions. The lectin chain is tailored to bind to a specific sugar on specific chains on the outside of cells. And once ricin is on there, it doesn't let go. Up to 100 million can latch onto a single cell. It's like a ninja clinging to the bottom of a truck as it zooms towards the enemy's lair. Or, better yet, 100 million ninjas. 

This is what 100 million ninjas looks like

The profuse binding of ricin to surface membranes allows internalization with all types of membrane invaginations. Experimental evidence points to ricin uptake in both clathrin-coated pits, as well as clathrin-independent pathways including caveolae andmacropinocytosis.^[18][19]  

Cells are constantly pulling bits of the surface membrane into the cell for a bunch of different reasons; signaling, protein recycling, even for digesting bacteria. With the help of certain proteins, the membrane buckles in like a balloon being poked, and then the hole closes in on itself and a bubble of membrane (called a vesicle) floats into the cytosol, the inside of the cell. Anything sitting on that surface gets sucked in with the vesicle...including ricin.

It's an amazingly simple yet elegant way to get inside the cell. It's like the ninja who hops inside the big basket of laundry as it is rolled, right into his adversary's HQ. Why blast into the cell when you can get the cell to carry you in?

Vesicles shuttle ricin to endosomes that are delivered to the Golgi apparatus. The active acidification of endosomes are thought to have little effect on the functional properties of ricin. Because ricin is stable over a wide pH range, degradation in endosomes or lysosomes offer little or no protection against ricin.^[20]

But there's a catch: the vesicle gets transferred into lysosomes, the demolition crew of the cell. These organelles break down all kinds of molecules. One of the ways it does this is by making the inside very acidic: most proteins, when exposed to the inside of a lysosome, would get more tangled up then last years christmas lights, eliminating their ability to do just about anything. How does ricin deal? It just sucks it up and powers through. It stays stable, in the same configuration, across a whole range of pH (level of acidity). This is unbelievably badass. Imagine a ninja that rode in with the laundry, got tossed in an industrial washer/dryer, went through a 2-hr cycle then leapt out, unscathed. THAT is what ricin does.

The perfect plan
(Photo by Ovi Gherman via Flickr)

Ricin molecules are thought to followretrograde transport via early endosomes, the trans-Golgi network, and the Golgi to enter the lumen of the endoplasmic reticulum(ER).^[21]

Once again, the cell's machinery delivers the ninja ricin to right where it wants to go, the endoplasmic reticulum: where the ribosmes hang out. 

For ricin to function cytotoxically, RTA must be reductively cleaved from RTB in order to release a steric block of the RTA active site. This process is catalysed by the protein PDI (protein disulphide isomerase) that resides in the lumen of the ER.^[22] 

Ricin can't do its job inactivating the ribosomes until it cuts the lectin chain loose; it's too bulky otherwise. But ricin couldn't smuggle a switchblade into the cell. It has to use it's MacGuyver skills and find a way to free itself from its lesser half. That's when PDI, the cells own protein, shows up. What does PDI do? It cuts disulphide bonds. It's an essential part of helping proteins manufacturing inside the endoplasmic reticulum. Guess what kind of bond holds together the two parts of ricin? You guessed it, disulfide bonds. Ricin uses this makeshift blade to cut off it's now-useless limb, 127 Hours-style.

Free RTA in the ER lumen then partially unfolds and partially buries into the ER membrane, where it is thought to mimic a misfolded membrane-associated protein.^[23] Roles for the ER chaperones GRP94 ^[24] and EDEM ^[25] have been proposed prior to the 'dislocation' of RTA from the ER lumen to the cytosol in a manner that utilizes components of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. ERAD normally removes misfolded ER proteins to the cytosol for their destruction by cytosolic proteasomes. Dislocation of RTA requires ER membrane-integral E3 ubiquitin ligase complexes,^[26] but RTA avoids the ubiquitination that usually occurs with ERAD substrates because of its low content of lysine residues, which are the usual attachment sites for ubiquitin.^[27] Thus RTA avoids the usual fate of dislocated proteins (destruction that is mediated by targeting ubiquitinylated proteins to the cytosolic proteasomes). In the mammalian cell cytosol, RTA then undergoes triage by cytosolic molecular chaperones that results in its folding to a catalytic conformation ^[28] that de-purinates ribosomes, thus halting protein synthesis.

At this point, ricin is sitting on the inside of the ER, like a ninja crawling through the air ducts. The ribosomes are sitting unawares on the outside of ER (to be fair, all proteins lack consciousness and are therefore unawares). To get into position, it once again tricks  the cell into doing all the work. It buries itself in the ER membrane so it looks like a mis-folded protein. The cell recognizes that, and flips the protein over to the outside of the ER so that it can be destroyed. Normally, it would get tagged with ubiquitin, the cells universal symbol for "dead man walking". But the ninja ricin is too slippery; it doesn't have enough of the lysine residues that are used to attach ubuquitin. Now, ricin looks like a normal protein. The cell has dropped ricin smack dab in the middle of a sea of ribosomes, but it still isn't ready to strike until cell chaperones, which help proteins fold into the right shape, descend on what's left of the ricin molecule, molding it into the perfect killing machine. The cell has essentially offered to let ninja ricin borrow its collection of throwing stars, just for fun.

Ribosome inactivationStudy of the N-glycosidase activity of ricin was pioneered by Endo and Tsurugi^[29] who showed that RTA cleaves a glycosidic bond within the large rRNA of the 60S subunit of eukaryotic ribosomes. They subsequently showed RTA specifically and irreversibly hydrolyses the N-glycosidic bond of the adenine residue at position 4324 (A4324) within the 28S rRNA, but leaves thephosphodiester backbone of the RNA intact.^[30] The ricin targets A4324 that is contained in a highly conserved sequence of 12nucleotides universally found in eukaryotic ribosomes. The sequence, 5’-AGUACGAGAGGA-3’, termed the sarcin-ricin loop, is important in binding elongation factors during protein synthesis.^[31] The depurination event rapidly and completely inactivates the ribosome, resulting in toxicity from inhibited protein synthesis. A single RTA molecule in the cytosol is capable of depurinating approximately 1500 ribosomes per minute.

Finally, ricin arrives at its prey, the ribosomes. Does ricin bust a protein-sized hole through where the ribosome used to be? No, that wouldn't be very ninja-like. Ricin reaches into the protein, finds a single bond in one of the RNA chains that ribosomes need to work, and slices it. Like a ninja nerve pinch (that's a thing, right?) this little cut totally inactivates a gigantic ribosome containing thousands and thousands of bonds. But ricin doesn't stop there. A single molecule of the stuff can inactivate "1500 ribosomes per minute". That's 25 per second. Beat that, Ra's al Ghul!

He can't beat that.
(Photo by Chaoss Control via Flickr)

What is responsible for ricin's extraordinary ability to infiltrate and destroy the cell from within? The short answer is, evolution. Most plant toxins (including those in recreational drugs like marijuana and cocaine) evolved to kill or badly hurt anything which tried to eat a given plant. The more effective the poison, the less likely the plant would be bothered by that particular threat again. The castor oil plant produces ricin in its seeds so that animals won't ingest them, thus preventing the seeds to develop. Over millions of years of plant-animal interactions, the protein has slowly adapted to take advantage of a laundry list of cellular mechanisms, allowing it to sneak in and sabotage the cells of unlucky herbivores. While other poisonous chemicals (like lead or agent orange, for instance) would, due to the shared chemistry and structures of animal and plant cells, be poisonous to the seeds as well as their consumers, ricin is specifically tailored to attack animal cells. It is the perfect assassin. 


The moral of the story: don't eat castor beans.