Rui Sousa, PhD, Professor of Biochemistry at The University of Texas Health Science Center at San Antonio talks about using a jellyfish protein that emits light to study transformation of normal cells into cancerous cells.
Interview conducted by Ivanhoe Broadcast News in October 2017.
In about thirty seconds tell us what you all are doing.
Sousa: We are building a molecule, a sensor that we can put inside cells. This sensor emits light because one of the components of the sensor is a protein from a fluorescent jellyfish. The light that this sensor emits changes depending on what the concentration of GTP is in its environment. The reason it changes is because we took this protein from jellyfish that emits light and we connected this protein genetically to this protein which binds GTP. GTP stands for Guanosine Triphosphate and it’s a master regulator that regulates all types of cellular metabolic processes. These processes include cell motility, division, growth, transformation status, and transformation from normal cells to cancer cells. All of these processes are regulated by Guanosine Triphosphate through its effects on G-proteins.
Where is GTP?
Sousa: It’s everywhere in our cells. People say ATP is the energy currency of the cell; well, GTP is also the energy currency of the cell. If you look at the amounts, you’d probably say maybe about in about seventy to eighty percent of the transactions, ATP is what’s used. But in about twenty percent of them, GTP is what’s used. GTP is more critical in regulatory aspects. Especially in controlling processes that change the shape of cells and that make them migrate and, in cancer, metastasize.
You couldn’t see it?
Sousa: We couldn’t see it [the GTP]because it’s an invisible, microscopically small molecule.
And so you borrowed some light if you will?
Sousa: We borrowed some light, we took this jellyfish protein that emits light and we took this other protein from a bacterium that binds the GTP. There are lots of proteins out there that bind GTP because they’re regulated by it, and we connected these two. And when this protein binds GTP, the GTP changes the shape of this protein a little bit. That’s quite common; it’s often seen that when proteins bind small molecules, they kind of wrap around them, change shape a little bit. But what happens is when one protein changes shape we can’t see that because of course these things are submicroscopic. But we’ve attached the protein that changes shape a little bit when it binds GTP to the jellyfish protein that emits light. And when the GTP-binding protein changes shape, it causes the attached jellyfish protein to change shape a little as well. And that causes the light that the jellyfish protein binds to change, as well. So connecting these two is a way to make the binding of GTP become visible as a light signal.
And what does that mean in the long-run?
Sousa: Well it means that we can see now see the GTP in the cell. For example, here are cells, images of cells, in which we put these sensors. And the more yellow, the orange and bright the colors that you see here are indicating to us that in those regions of these cells the GTP concentration is high. So GTP-activated processes like cell growth, motility may be more active in those regions, but we’re still beginning to explore this because we’re just starting to use these sensors. But you can see that for example over here in these images these processes extending from the cells are quite bright and this edge of this cell over here is lit up. And these may be regions where the cells are reaching up and growing and moving. So the high concentrations of GTP in those regions are activating those processes. That currently is our hypothesis, but that’s why we built this sensor to be able to look at this.
Meaning that this GTP could be a giant regulator so it changes and impacts the rest of the cells?
Sousa: Exactly, and it impacts these cells in what they do. So we see that there’s some regions even within a cell where there appears to be high GTP levels and GTP activated processes like growth and motility are active, and in other regions GTP levels are low and these processes are quiescent. And what we’ve done here in these images is we’ve added drugs, mycophenolic acid in this case. Mycophenolic acid is a drug that inhibits enzymes involved in GTP synthesis. So we expect that the GTP levels will go down and we can measure that chemically, as well as using the sensor to confirm that’s the case. And you can see what happens as the effect of the drug occurs in these cells, we see the bright colors disappear, and the cells just become just all blue and kind of dull purple, which tells us that the drug has worked and reduced the GTP levels in these cells.
The GTP has say begun the long slide into cancerous tumor for example, and you put a drug in there with it that would calm it down?
Sousa: Yeah, one of the things that our collaborator has found out, we found out, is that when melanoma cells become metastatic they increase the levels of their GTP synthesis enzymes. So the high GTP level in metastatic melanoma cells are essential for making those cells migrate, to make them metastatic. And he finds that when he treats these metastatic melanoma cells with this drug with MPA they stop migrating. In fact, if he treats them enough they stop proliferating, they essentially stop becoming cancerous. This is why we are focusing on the idea of these filopodia and cells becoming mobile, because of the idea that high GTP levels in certain regions of the cell are important to make cell migrate and to make cancer metastatic.
That’s important.
Sousa: It is important. I mean it’s good not to get too excited right away because we can find lots of small molecules that do this in tissue culture cell systems with cancer cells. We can identify lots of compounds that will inhibit metastases, inhibit growth, and inhibit the cancer cell penetration of different organs. Those compounds are the important ones, they’re the ones we want to focus on but we may find hundreds or thousands of those. But then we have we go to other levels of testing: OK, they work in a dish with cells; do they work in mice? do they work in humans? do they have side effects? That’s a very long way to go before you actually get to a drug.
But what are you hoping to do with that, are you hoping to test certain drugs against the GTP or you are hoping to stop the metastases of cancer?
Sousa: We’re hoping to use them to identify new compounds that reduce GTP synthesis in cells. One of the ways that we can do this, and this is the point at which Dr. Hart can show us his equipment, I mean you can imagine we have a library here in our drug screening facility of a hundred and fifty thousand compounds. Well, it would be impossible to say take some cells in a dish, add one of these compounds and then say three or four hours later check the cells to see chemically by putting them in a machine, it’s called HPLC, to see if they have less GTP. That would just take forever. But another way that we can do that if we have this type of sensor is we can just grow cells in these dishes that have thousands of little wells, sometimes tens of thousands in a single dish or at least thousands in a single dish. And we can put these sensors inside those cells. Then we can add to each well different compounds, and we can just go and look very quickly using these screening microscopes that Dr. Hart will show you and these computer images and we just very quickly see whether the GTP level has gone down or not.
Where do you get this green light from the jellyfish?
Dr. Sousa: The jellyfish protein itself emits light. Like in the movie Avatar, you have this planet where the organisms glow with light. Well, that was inspired by something very real here on earth: there are lots of organisms; especially jellyfish and anemones, which make proteins that glow, that naturally emit light.
So you just take the protein out of the jellyfish?
Sousa: No we don’t take the protein; we take the DNA from the jellyfish. All of the manipulations are done at the DNA level because that’s where you can actually do it. The jellyfish make a protein that emits light called green florescent protein. Somewhere in the jellyfish’s genome there’s going to be a gene that encodes that protein. You take that gene and connect it the DNA for the protein that binds the GTP. Then we take that DNA, which now contains two fused genes, and put it into the cells, let’s say the cancer cells, you want to study. Those cells make protein from the DNA, and now you have the sensor in the cells.
I hear this doctor and want to know, what does that mean to me?
Sousa: It means that probably in the near to medium term, honestly not a great deal. I personally am more a basic scientist interested in the biology of these processes. What I find fascinating when I look at these images is the fact that I see this bright edge here. That suggests that there’s a very high GTP concentration in this region. I want to know how is that GTP concentration achieved there, how is it sustained, what is the mechanism of it and what is it, what is its affects on the biology itself? But folks at home, I can’t get that much more compact than saying well, what we are doing, what’s relevant for you is that you will see technological examples like this, this one possibly as well as others, used to screen for new cancer drug leads, that’s true. That’s something we’re writing in the grant right now, that’s something that we’re actually doing. But quite likely these types of technologies will become more prevalent for looking at other phenomena inside human beings. But you know honestly that is a difficult, the proper application, really I don’t think going beyond the idea of screening for drugs is about it for now. In addition to screening for drugs with cells, we can also use this for screening with purified systems. But basically the immediate answer for the viewer watching at home would be we can use it to screen for new cancer drugs that operate through this mechanism, through effects on GTP levels.
How do you get the drug in that?
Sousa: The drugs that we will be screening for are simply added to the solution that the cells are growing in. And we rely in most cases on just passive diffusion of the drug into the cell. That’s still how you know for most chemotherapeutic agents. I mean you get them into the person by intravenous methods, sometimes orally. But then you just rely on them passively dissolving in the bloodstream and then penetrating the cell membrane. There are newer drugs that are actually directed towards certain receptors but I’m not enormously familiar with that.
END OF INTERVIEW
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