Poisonous Snails and Our Cellular Membranes: Kallol Gupta Makes the Connection

Richard: Hello, this is Richard Jacobs with the Finding Genius podcast. My guest today is Kallol Gupta is an assistant professor of cell biology, is his own lab, Kallol Gupta lab at Yale and we’re going to talk about these deep-sea snails that you studying off the coast of India and the whole world of biology. Thanks for coming.

Kallol: Thank you very much for the invite. I’m really excited.

Richard: So what got you interested in these particular snails? Why them?

Kallol: Yeah, so it was rather an accident that I chanced upon them like many students, or at least I should speak with just myself. I have very little idea about what it is before I started working, so I thought I was a physical campus before I started doing that and then my supervisor said that just because you have done something for money at this point, that you have to be doing it for the rest of your life and that really inspired me. So I don’t even like the idea about looking at that’s what I because these peptides all bind to the different genes and receptors, which is what I’m currently working with and the idea is these days are all neurotoxic. So somewhere there they are basically perturbing your cellular physiology. So whatever immobilizes you in a regulated dose, that’s basically an anesthetic. So the idea of the motivation behind studying these many others, actually, which is active in nature, is to basically understand how these organisms in biology have fine-tuned their arsenal of toxin so they can actually specifically target different channels and receptors in the central nervous system and that really hijacked our physiology and that sounded like a very interesting question and it’s interesting because my first thought was basically to call the time, I would say, into the deep sea to with the fishermen to get the snails and then get them back in the lab, sacrifice them, get the toxins, and then basically a very rich concoction, a few hundred polypeptide molecules.

Richard: What do you know of the natural environment of these snails like who tries to grab them and who are they defending themselves against?

Kallol: Actually even in the same natural environment, different kinds of snails based on their feeding habits would have a different set of toxins and this is a perfect example of evolution. So that’s true based on their feeding habits, it would actually create a different set of toxins or a mixture of toxins which are fine-tuned to that kind of thing that they’re trying to target so that the natural habitat, as well as the feeding habit, is actually very, very critical. If one kind of snail, which has no particular feeding habit and keep it in a controlled environment, a laboratory environment where you keep feeding, let’s say a snail, you start giving it fish so does it actually change the venom that is produced in within the lifecycle.

Richard: What do these snails eat normally?

Kallol: Fish and it’s actually really interesting because the first example of these snails in modern literature comes from a World War diary of an American soldier who was actually posted in the coast of Japan in the Pacific coast and then he encounters these snails and that he writes in his diaries and then somehow it’s all forgotten. Somewhere around 1990 and mid-90s, Baltimore Olivera for the University of Utah, who was at that point started working with these pharmacologists, a molecular pharmacologist and he started showing that, look, these things are basically producing a concoction of molecular libraries, which have no kind of evolutionary fine-tuned to look at a different membrane, proteins, and receptors and hijack our biology.

Richard: So what are the snail venom’s target. Are they different from, let’s say, a cobra that would bite you?

Kallol: So, yes, they’re different. So I also partly worked with snake venom during my Ph.D. and in some way a bit boring because they both have they all have mostly what they call alpha-toxin combined to a particular receptor called the nicotinic receptors. It is the receptor that is also responsible for acetylcholine. It’s a central receptor in our central nervous system and not detection of pain perception. This is also the receptor that gets impaired upon smoking. So it’s a big pharmacological target for smoking cessation and so what it does, basically, it blocks your perception and this is how I got interested in memory because in the first part of my Ph.D., I was rather an ignorant Ph.D. student whose idea was just to sequence develop mass spectrometry methods. You just sequence these proteins to know their structures because these venoms that are produced know the structure, the architecture of these and developing new mass spectrometry, that was my focus. But we end up in civil war broke out and as a result of that, it was to the deep sea, like the fish. They stopped going to the deep sea because that’s very close to Sri Lanka and they’ll get shelled by the Sri Lankan army. So as a result of that, I stopped getting samples. So I remember I woke up, I walked up to my supervisor and I asked exactly the same question that you asked, that rather than mining what we said, then he said that, OK, since you’re not getting any sample, why didn’t you meet this question? You will rest of your body and then I started looking at stuff that I discovered where they find for me was some of the receptors were already known. But some of the novel toxins that I discovered, we found out where they minor. They typically bind membrane through things that are basically at the cellular level. So if you if a comparison with a just like a room has a wall, the cell has its own one and just like a room has a as a door or window through which you can communicate with the surrounding cell also has its specific doorways and windows, which are mostly made of a specific set of proteins, because they reside at the membrane now 60 percent of the drugs that are currently available at the market, that’s how much important they are to regulate our cellular physiology and different pathophysiological conditions from cancer to different forms of cancer to new innovation. You would see many proteins cropping up again and again even cause different physiological information. So these toxins and basically bind to these cells, membrane proteins whose job is basically to propagate neuronal signaling. So if you think about perception, which is called scientifically cardinals deception, deception is just neutral signaling, a particular kind of neutral in a few minutes, which leads to reaching out, which are read by a specific set of people. Now potassium channel, you could drink receptors have evolved to generate a large area of molecules, it just throws everything at you. But even if, let’s say, 10 percent of it works in your body, that’s nearly successful and it has been successful in blocking the pain perception in your physiology.

Richard: Why would it mask you from feeling pain?

Kallol: So most of these things are not deleterious to a human being simply because of the size proportion. So they can just deliver that much of toxins that we need to create a human being and this is the green line. Anything that is making you unconscious in a regulated dose that is a painkiller, it’s exactly the same thing. Just the dosage is regulated.

Richard: So if you were able to harness these snail’s venom, you could, I guess, have multiple levels. You can maybe do a painkiller with it. You can take it further and do anesthesia. So you take it further too.

Kallol: Yes. So that’s precisely why these and many other natural peptide-like these are very, very important because that’s exactly what we ought to do and one of the key reasons, the motivation behind this is exactly what I was doing before that six percent of the drugs are targeting membrane proteins where we had declining structures using imaging approaches like structures of proteins have become much easier to, in specific, have become much easier than, what, even five years back. It’s a question that comes that standing in front of us is we specifically target one set of tools. We are being very closely related analogs, and that’s where these things have figured it up. It’s figured it out through a temporary window of evolution, but they can have fine-tuned the genomic library to target only one set of blueprints, but not all that. I can give you another example of walls, but they basically they would immobilize their prey not to feed on them so that we organize their prey so that actually they can lay an egg inside and that now just immobilized, but it’s still alive and now that body is basically working as an incubator for that was to actually take and once the eggs are all hatch, then there’s no need of the whole cell. So to the newborns and basically dropped out of these organisms which are immobilized by the vent. So beautiful chemistry or biology, whatever you call it, which they have figured out over eight years of evolution. That’s really fascinating and one of the reasons why we’re interested in studying and another way is to look at and figure out how other organisms in the future have already done and learn.

Richard: So in a way, the snails are kind of like a parasitic wasp, in fact, a creature stunted or incapacitated, but it’s still alive and they lay eggs in eggs and I guess when the eggs actually eat their way out or dead by then or what happens.

Kallol: Yes. So the snails don’t do that. But there are certain kinds of wasp that would do that.

Richard: this reminds me, I had a fish tank years ago. I remember they were fish it and all of a sudden the snails appeared and where they came from and then I saw one day at a fish was dead and there were two snails separate onto it and I thought how they at the snails catch. Snails barely move and the fishes swimming around. So maybe the same thing happened in my little fish tank years ago that you’re talking about.

Kallol: I would be surprised if you have seen snails appearing in your fish tank, whatever it is, it is definitely in the same light that we have co-evolution has basically put tremendous stress on these organisms to live in an extremely hostile environment with the slow speed to develop a unique arsenal which separates them from the rest of the food chain and it’s beautiful chemistry to start in thinking about the shape of a snail and how it’s so slow compared to a fish. So they mean the fish is mouthing the snail shell and that’s how it gets affected.

Richard: I can’t see how the snail would get into a position where its flesh is attached to the fish or maybe I guess the fish starts to swallow it then. Maybe it’s in venom. What are the different mechanisms that people have found out that snails can venomate a fish?

Kallol: Yes. So for the snails they have actually many of them have developed mechanisms. So just like throwing an ax, they basically have been intense where the synthesized them and it’s stored in the venom that they get that then I want to bring it basically fills that and let the venom throw it like an ax and it just stings the pre and the moment it does that, it’s immobilized.

Richard: you mean when they see a fish, they spit at it?

Kallol: Absolutely. In the majority of the cases and then they swallow everything and then they’ll keep it in that environment inside the mouth, whether it’s a higher percentage of toxins which will eventually sedate all of the fish, just swallow all of them. So the different mechanisms, but the harpoon mechanism is the most common one.

Richard: But in the water, how could a harpoon work but I would think this looks like a cloud of venom kind of floats around it. The fish swims through it or you think there are harpoons available underwater?

Kallol: There are videos available. If you’re interested, I’ll send you some. It’s beautiful. Of course, there’s more resistance inside water. So but they use all the muscles to overcome that resistance

Richard: I am surprised about in the water is picturing a little harpoon because the water changing its path, like, how could you get a straight path? How would you target anything even a few inches away in the water? What is the harpoon itself look like? Is it composed of a liquid of a higher density or is it physical?

Kallol: No, no. It’s a tissue-based organ and inside that, you have the venom, which is basically like a needle that is injected onto the fish.

Richard: Like a pseudopod or something.

Kallol: Absolutely. Yes. You’re right. That’s why these creatures sometimes would be in the deep, especially due to human activities compared to the shallow end of the sea. You have to go quite a depth to before you can actually see.

Richard: In your analysis, then, what do you think will be the most useful thing that will come out of you studying the snails? Will it be a new painkiller, no anesthetic or just understanding of how this works or what?

Kallol: Yes. So what I described is something that I used to do in my research has progressed a bit. What happened when I started understanding how these work on memory improvements. I think he got interested in memory, so after I finished my PhD when I grew up with Microsoft, that’s when I started getting fascinated by it. So it’s kind of two sides of the story and my dad is studying these molecules, which both have some interesting stuff. I’m not even studying those instruments which were the target of these molecules and how they assembled in over the last one and a half year period, set up our lab. We are very interested in bringing them together. So one of the key aspects of studying proteins now is to look at how these proteins are interacting with each other in the membrane, how these proteins are assembled in the membrane, what kind of are molecules they’re interacting with and how can we study them directly from the membrane environment? The cellular membrane environment is a very, very challenging environment for any biophysicist, simply because there’s a lot of liquid water-soluble and it has been a medium to challenge, to study anything that is embedded in the membrane. So what we are really interested in doing now is to develop approaches using mass spectrometry, which is in some way a glorified we need to study these macromolecules protein complexes sitting on the membrane and then we use the molecular resolution of mass spectrometers to understand what kind of other molecules binding to these proteins, what happens when these molecules want, what associate with these groups and basically looking at the molecular orchestration of the signaling proteins which are regulating a large number of cellular responses directly from the membrane environment and how external stimuli are manipulating its signaling gaps which are taking place in our cell every move in a different life. One of the key aspects that we are really interested in studying is to look at can we ask the question that can lead to a set of improvements which are maybe responsible for pain perception, may be responsible for some of the growth events, but they are deformed due to particular disease conditions that we know. Ask the question that in a particular disease condition, what are the molecules it’s interacting with? What happens if I throw another set of possible molecules that and then what happens to that cellular? Did this the native cellular organization? Now people are interested in these sets of proteins, which are really important for a large number of functional reasons, enough etiology. What we’re really keen on looking at is how these molecules are assembled in the membrane. What happens if you pull these large number of toxins? Doesn’t that in biology, how they interact with each other? What happens to the native cellular organization in the presence of these 500 to 1000 toxins present? A single challenge here is a snail would produce little five hundred peptides or that’s 500 toxin molecules. So to be so out of these 500, maybe only one or two of binding to your target protein of interest, how many people wonder, how do you pick that one or two of that 500? So there we are now using the power of nuclear power, of mass atrocities so that it can actually impact what kind of molecules I think of it so that we can now do whatever I did, let’s say, 10 years back in a blind manner where I would start finding out the sequences and the structures of all the toxin molecules present, the kind of guesstimate based on the structure, which one might find where we are actually going to step ahead because now we can actually directly look at these proteins and the receptors where these receptors from the cellular environment with all these toxins and see which one might read what happens when they buy and these kinds of questions would be for two reasons. One, it gives us a unique molecule airport to understand cellular physiology for many of these proteins. We don’t really know-how. They don’t mean cellular cascades. That’s what these natural toxins give us, a fantastic molecule which doesn’t get exactly one protein of interest every second. We can actually use this platform as a large scale drug discovery platform where you can activate the natural toxin libraries and concoctions of hundreds and hundreds of proteins and then specifically asked the question, which one binds to it, this would give us a fantastic height dissect the natural libraries of boxes in a very meaningful manner within a very short period of time. This is part of ours.

Richard: That’s a great idea. Why not get a couple of fish tanks and find out which fish that these snails prey on most often and have them do it in a tank and then take the fish before it dies at various stages and they look at it and see where the toxins are going with tissues they’re preferentially attacking with cell types. That would give you a good scorecard for your analysis to see if it’s working.

Kallol: Yeah, so that’s exactly what I’ve been doing. But instead of doing it on a fish, you can do it in a much more targeted. One of the problems of doing it directly on the fish is that you have on both ends you’re looking at a large number of possible hits. Just in human, we have almost 20 percent of our genes. Almost 25 percent of our express proteins are membrane proteins. So looking at proteins would be fine. So it’s a thousand across 500 problems instead. You can see that. Look, I know this particular amendatory is really important for breast cancer. I want to see in this set of toxins in the snail, which produces 500 peptides, is there anything that binds to this protein? If it does, which one binds want to structure? Where does it bind? This information would know in less to directly target to a targeted molecule and discovery against a protein.

Richard: OK, so and then what is the end goal? What do you want to understand how they interface with the membranes?

Kallol: So one of the two interests in this one is of fundamental interest in understanding memory, protein biology is and the methods that we are developing and the platforms that we are developing now, enabling us to see things that we could be of, to look at processes that we could not do before. So is a fundamental change in the field that can we look at all these proteins that are organized in the membrane. What happens in the presence of different chemical proteins? What happens to the organization’s functions and structure-function? So that’s a fundamental problem which we are very interested in understanding in the lab to develop. It needs a new experimental approaches using which we can address this in a wide number of systems. But then there’s another part of the lab which is interested in studying in a much more focused on where we have one or two pieces of groupings where we are trying to understand that which kind of molecule could point, which are the critical drug targets that well, and there are a large number of proteins where the drug targets, we don’t even know what’s a good topology of a protein to target. It’s a good binding target when you’re developing drugs, so if we want this time to do it, we can probably then address the question in a completely different way. In a way, we would then mimic nature instead of doing it from scratch. So that’s how I use the commercial interests of other, I would say, a practical interest, which we think can lead to a deliverable molecule or at least a deliverable idea which can lead to a molecule that can be used in a medical system to know to alleviate different physiological conditions.

Richard: Do you have any insight yet on which of these hundreds of peptides seem to be predominantly active and which are just there?

Kallol: So the interesting thing here is everything has a function and that is the most enigmatic part of this question, that there are 500 molecules you can think about in these organisms are under tremendous evolutionary pressure. If there is anything that is of no use, they would have to it out from their genome by now. The reason for it it’s not filtered out is because it serves a purpose. Now, what’s really interesting is that we don’t really know what are these kinds of toxins and that’s large because we don’t like what we do a molecular discovery of these toxins and where the mind and the experimental methods that we are developing now, we do we are going to be there very soon. We can actually ask these questions in a much more height manner and that’s what we need to understand because I believe that they are producing something which is of no use to them.

Richard: So what are you looking to do? Are you looking at the structure of the proteins? Is that a technology that’s yet available to determine structure?

Kallol: Yeah. So the structure of the technologies is now available and they have been available which has become much easier. But in the short term, the determination process is very low. It also requires a large number of knowledge. Among others we’re developing in the lab is not much which is basically not allowing us to look at protein complex. But at the same time, we could look at a small drug molecule of a few hundred dollars, a few atoms. So that threat of the resolution that is now enabling us to look at these large assemblies of the proteins, but at the same point of time, see which molecules are binding on top of that now, developing a process where we can update these drug-protein complexes breaking down inside the mass spectrometer and find out what exactly is this drug, what is exactly the polypeptide sequence of this drug, but that doesn’t really solve the question because we don’t really know. We can’t really predict by looking at that structure which of these 500 peptides point at that’s limiting the resolution of the mass spectrometer is really, really critical because it gives you unambiguous identification of which are the molecules that are pointing to your protein binding, how many are binding, in fact, if there are any special modifications upon binding and you can look at all of that now from a very preliminary physiological environment of the cell membrane itself and that’s what happens in the lab.

Richard: But in mass spectrometry, can you cut a giant protein? How do you figure this out with a gigantic protein?

Kallol: Yes. So mass spectrometry we do. It’s slightly different from typical mass spectrometry, which probably many of many, many people around the world uses, which is called proteomics that many people are familiar with and typically the typical proteomics I use to find is the structure of the protein. So you either need to create by adding organic solvent or you add to proteins with an up the properties of the protein into smaller bits. So the mass spectrometer is built in the ion optics as well as the front, as well as some of the pressure readings of the mass. We promised a fine-tuned. Look at the intact protein complexes going up to as big as the entire ribosome enables us to look at the large protein complexes, the structural integrity. Now, while we are doing this, the resolution is also very high. So why do we make a protein complex? So that gives us the confidence of what exactly is on top of that. We have now developed approaches in the lab, which is not enabling us to take these protein complexes directly from a cellular membrane-like environment. So the physiological condition and this has been a major, major challenge in recruiting field because I want to get them out of the membrane in our water-soluble to unlike most proteins, they’re not stable. We have figured out that we can now detect these things directly from the cellular environment itself or cellular membrane-like environment. On top of that, we have now developed a way where we can now fragment these molecules, these gigantic protein complexes with ligand mounted, with the molecule bone directly inside the mass spectrometer and just like doing a lot of sequencing now you can see which of the molecules binding to it. What is that chemical structure? In fact, we are in the process of developing ways where we can even retain the structural information of which region of the protein where this particular molecule.

Richard: What have you discovered anything unusual or interesting, particular peptides?

Kallol: That’s one of the key things that are remarkable, what’s fascinating to see, though, is how not notice which side finds the way, but how that finding is modulated by another layer of regulation of the lipid molecules and that’s really fascinating as we have been working with a couple of transporters which are responsible for not exception and we can see that that is specifically inspired with their specific abilities and it has been absolutely fascinating. It’s early days and just when we’re getting really, really exciting results, we got everything back to the lab very, very soon and it’s a fascinating time most days as it is and lab things don’t work as we want it to be. But at the end of the day to that process, we learn something about the stage now where we are now taking up some of these really challenging proteins to work with and trying to understand that what kind of peptides binding they are, what kind of molecules find out what’s the role of lipid as a cool factor in modulating these structure assemblies.

Richard: What would make a protein hard to work with versus not?

Kallol: Let me answer that from the perspective of membrane protein. What makes it harder for a member to do so? Because proteins are in a very unique environment, are residing at a cellular membrane which is completely devoid of water, mosquito bite of water. So unlike most other proteins, so 70 percent of a protein, you can purify them and put it in a solvent and then do your work with that amendment. You can’t do that at the moment. You strip them of that hydrophobic but from that hydrophobic lipid, they would lose their structure and are not functional at that point. So there’s no meaningful from. A study you can do with them at that point, so the challenge is how do you keep them out of there? So if you keep them out of their environment, but at the same time keep them and people have been working with lots of membranes due to basically mimic the cellular membrane conditions. What we figured out is a way we can now look at things directly from the membrane-like it. That’s where I’m aspect set up that we have in the lab is very, very unique because it now enables us to get rid of this idea of using membrane mimicking because even the best is limited and we don’t really know when it’s not doing a good job.

Richard: Yeah, it’s very cool. We’re almost out of time. What’s the best way for people to find out more about your lab’s work?

Kallol: Yeah, just look at our website or just write to us. We put everything we put things in the archive also before publishing anywhere so that it’s open to the public and anybody can read it because that’s actually really important for the propagation of scientific ideas. It comes from the idea of free speech and free knowledge. So it must be available for people to read all of the scientific journals.

Richard: Very good work. Thanks for coming on the podcast.