The tiny world of peptides

CHRIS HATZIS
Eavesdrop on Experts, a podcast about stories of inspiration and insights. It’s where expert types obsess, confess and profess. I’m Chris Hatzis, let’s eavesdrop on experts changing the world - one lecture, one experiment, one interview at a time.

We see the word 'peptides' present in many things. A lot of health and cosmetic products contain different peptides for many uses, such as their potential anti-aging, anti-inflammatory, or muscle building properties. But what actually are peptides and what else can they do?

Dr Troy Attard is a part-time application specialist in the Melbourne Protein Characterisation
platform at the Bio21 Institute, University of Melbourne. His specialty is peptide synthesis. Troy is also a senior scientist at Mimotopes, a company that supplies peptides and related materials to research and industry. As if that wasn’t enough, Troy is also a first-year chemistry tutor at three different University-affiliated colleges, where he teaches several chemistry subjects.

Dr Andi Horvath sat down for a Zoom chat with peptide expert Dr Troy Attard.

ANDI HORVATH
Troy, you're in charge of a peptide lab, what are peptides?

TROY ATTARD
They're basically short proteins, which are chains of amino acids that are joined head to tail, a little bit like links in a chain. That's pretty much what a peptide is.

ANDI HORVATH
I know proteins are involved in every cell of our body, they're from metabolic to communication to how things interact in our body, so why are peptides important?

TROY ATTARD

Well peptide research is important because they make that study of proteins, or they facilitate that study because peptides can be thought of as fragments of the protein. You can identify the important part of the protein that might bind to a receptor, or it might bind to another protein, and thereby switch it on. You can identify that important little part of the segment and you can synthesise it as a peptide because actually isolating proteins is quite an involved process, and sometimes getting access to the proteins that you're interested in can be quite difficult. It can be more convenient to make the segment of the protein, the interesting bit, the active bit if you like. That's how the peptide fields have emerged.

Of course, peptides also are very interesting molecules in their own right, and there are what you would call peptides that are natural in the body as well. Insulin is an example of a peptide, it's two peptide chains that are joined by a couple of bridges. There are a lot of small proteins that you would consider peptides and they do – well they have all manner of functions in the body. You can manipulate them for whatever purpose you'd like, such as just basic research if you want to investigate what parts of a protein are involved, you can make a whole range of peptides that represent that area, and you can assay them and test which ones are important for binding, whatever that binding partner might be. Then you can play around with the sequence, you can mutate different parts of that peptide to see which parts of that segment are most important to the protein.

Just by doing this basic research, you can build up a profile, and you get more knowledge about the interactions that are going on in the cell. When you come across a problem such as the classic one is cancer of course, the more you know about that mechanism, the better a position you'll be in to develop a therapeutic, for example.

ANDI HORVATH
By modifying peptides or analysing them, you can make them stickier, you can make them coil more, you can make them target things, and that way you can control the action of peptides, have I got it right?

TROY ATTARD

Yes, that's right. They're very versatile, and the good thing about peptides too is that they're very specific for their target. You can join or you can attach a lot of things onto the peptides, because of their structure, it's like they've got a whole lot of little chemical hooks.

I'll give you an interesting example of some of the work that's going on here at Bio21. One or two groups are looking at - well they're looking at using a particular peptide that binds to a receptor, and in this particular cancer that they're looking at, the cancer over expresses that receptor on the surface of the cell, in other words it makes many, many copies, much more than a healthy cell. What this group is doing, they're attaching some of these groups to the peptide, so you know that the peptide when placed in the bloodstream will bind to these cancer cells, and what it's got attached with it is dragged along.

One example is imaging, so what you can do is you can attach a fluorescent group, or a chemical group that gives off a signal under a scan, it glows, it gives off a particular colour. When you attach it to that peptide and then place it in the body, that peptide will bind to the cancer cells. When you screen a patient, for example, you can look at the scan and you can see the bits that light up, you know the areas that have the high incidents of cancer cells, or the high density of cancer cells. You can use peptides as deliverers of cargo.

ANDI HORVATH
That's a great story. It's a transport mechanism as well.

TROY ATTARD
Yes, that's right. Not only for imaging, you can also make these little molecular cages that are just like what the term suggests, they're little cages with a cavity inside, and you can attach that to that peptide as well. Inside the cage you can place what's called a radionuclide, which are particular isotopes of different metals that give off radiation that will kill cells, but that radiation has only a short span, it's only dangerous within a small distance. When the peptide finds its cancer target and binds, associated with this peptide is this cell killing radionuclide, and of course it will destroy the cells in the immediate vicinity, which are the cancer cells. That's similar to the imaging approach.

ANDI HORVATH
Troy, give us a case study of where you modify or identify or build proteins, or rather peptides?



TROY ATTARD
Well there was one interesting project I was involved with several years ago when I was working for the dental science department. We were looking at antimicrobial peptides, and the background to that project was it all traced back to this little creature called an African clawed frog. It was found that if you make little cuts or lacerations on its skin, it seems a little bit mean, you'd hope it didn't hurt the frog, and then they placed it in an environment which is rich in bacteria like a dirty pond situation. It was able to not only not get infected, but it was able to heal itself. The agents that were identified that allowed it to do that were antimicrobial peptides that it secreted on its skin; it gave it a defence against the bacteria.

Looking at these peptides, the next thought would be how can we use – it’s a natural product, how can we use that for our purposes. I guess the bigger picture is trying to overcome antibiotic resistance and the emergence of superbugs. Peptides are thought of as a very promising alternative to antibiotics.

We made a series of these peptides and tested their activity against various bacteria, they're all oral pathogens because I was in the dental science department, you wanted to make sure that it was relevant to your area. Alongside this was also the knowledge that a lot of these peptides, they coil up, so they form almost like molecular springs if you like to think of it that way. It's that coiling that facilitated the killing of bacteria, because these peptides punch holes in the membrane of the bacteria, and basically the guts leak out. That's the mechanism by which they work.

The idea was maybe we can take these peptides and modify them to coil even more, and so that's what we did. We did find, there's various strategies that you can do that, we can add bits onto the peptide and see what they do. In a lot of cases we actually increased their potency against some of these bacteria. The only problem is from that point on is how do we make it specific to attacking the bacteria, because they also were very good at killing human cells.

Often when you find an answer in science, there's another three problems that you have to overcome. That was a very interesting project I was involved with.

ANDI HORVATH
Troy, tell us about how you got into the peptide area, and I'm keen to know what was one of the memorable case studies that you worked on when you first started in this area?

TROY ATTARD
I’ll change that question to how did I get into science. I have some interesting hobbies, and it was the love of nature that probably caused me to gravitate towards the science field, pardon the pun, because I have a large collection of carnivorous plants, and I've kept reptiles with pythons and tropical frogs and all sorts of things. It was just that love of nature that drew me to science.

Then I think from that point on, as in you see with a lot of cases, it's actually driven by opportunity. I didn't actually know exactly what I wanted to do. The opportunities that were presented to me just led me down this pathway, and I ended up – I went through the biochemistry avenue, and a component of those projects when I did honours was making peptides and testing them, what they did. Then from there I found a position where I started to specialise in making peptides and that's sort of where I've stayed from that point on.

I think maybe the antimicrobial peptide project was an example of one of the interesting projects. Another one was developing peptide vaccines, again in the dental science department. That's an emerging field, because peptides are seen as very promising candidates for vaccines because they're smaller, and they're most likely, or they're less likely I should say to elicit an allergic response.

Just thinking about details of that project, basically when you have an antigen that's invading the body, what the immune system sees is the surface of that antigen and the shape of it, and it will attack that. That's its recognition. You can take a large protein and you can just look at the peptides that make it up, the surface peptides for example, and find out which of those surface peptides really gets the attention of the immune system. Then you can take those peptides and create a vaccine construct just with the peptides that are components of the larger protein.

That's the basic idea behind it. That's still ongoing, I don't think there's a peptide vaccine on the market, I haven't checked for the last few years, but I think researchers are closing in on it.

ANDI HORVATH
Tell us about changes you've seen in the industry, what has really evolved in your area of peptide biochemistry?

TROY ATTARD

I'm probably more in a position to comment on the synthesis of peptides I think, because as I collaborate with various people I often just get little snippets of the biological story, and generally I've been involved in making the peptides that say biochemists or biologists require for their research. I think probably the biggest changes, or the more significant changes have been in the technology that allows you to assemble a peptide chain.

Just to put that into context. When I did my PhD, I manually assembled peptides as most people would have, and it wasn't really that long ago. It would take about an hour to an hour and a half to attach the next amino acid in the chain. If you were making a peptide that was about 20 amino acids long it could take you three or four days if you're working 9:00 to 5:00 to actually get that peptide.

These days it's done on a microwave synthesiser, so the temperatures used are a lot higher, like 75 or 90 degrees, and the chemistry is adjusted to cope with that heat. To put an amino acid onto the growing chain would take these days between two to four minutes. That 20 amino acid peptide doesn't take three or four days anymore, it takes maybe an hour and a half to two hours and you're done.

That's probably the most surprising change, or stark change that I've noticed over the years. Now we're relying on this sort of technology, it's very hard to go backwards. If you go into a lab that doesn't have one, it seems like you're going back into ancient times.

ANDI HORVATH
Troy, your kind of life the Lego master of peptides, you also work for industry, tell us about that.

TROY ATTARD
That's really focused on synthesis too, the industry part. A couple of days a week I'm working at Mimotopes, which are a peptide company, and they get all sorts of requests. I'm a senior scientist there, and so the jobs that come in, or the peptides that I help out with are the non-standard peptides where researchers have been quite creative in their requests. I sit down with a couple of people and we work out how we're going to put these peptides together. That's basically the other part of my job is purely focused on making, in an economical way, these challenging peptides.

ANDI HORVATH
Troy, you know the old adage you can't uncook a cake, or you can't unboil an egg, well you're the peptide king, so I'm dying to ask you the question - a few years ago science declared you can now uncook an egg. OK, give us some insight to how on earth that’s possible.

TROY ATTARD
Uncooking an egg, yes. I suppose if you think about the process of – it's actually boiling an egg, it's a slightly different process to cooking, it specifically has to be boiling an egg. What happens is the proteins inside that egg, they have their certain confirmation and they're in an ordered structure. When you boil that egg, because of the heat the proteins unfold, so they unfold into their linear confirmations, and it becomes like a tangled bowl of sticky spaghetti if you like. Once that's formed, which is the boiled egg situation, the egg white, it's very hard to – well up 'til this point in time, it had been considered as the adage said, you can't unboil an egg.

What that refers to is you can't retrieve those proteins back into their natural shape, but this group of researchers was able to do that, and this related to work that they were doing in the lab. They just used the egg as a difficult example in other words, it was like a challenge, and if they could demonstrate retrieving those tangled proteins or some of them, and isolating them back into their natural form, then that was proof of concept of this process.

That's what they did, and they did it using a vortex fluid device, which is like a spinning test tube. It seems quite simple when it's described, but all it really is is a spinning test tube and they had to measure various angles which is the best angle for spin, and that type of thing. But it's like a spinning test tube on its end. The egg solution, or the protein solution from the boiled egg would rise up the sides, and in doing so those proteins will be pulled apart from that tangled mess, and not only that in that environment they'll refold into their natural confirmation, into their natural shape, and then they're collected at the top of the tube.

Of course I should mention in that solution there were some denaturing, it was a denaturing solution, so it did help to lubricate the peptides somewhat. But that represented a big jump in the technology, because if you have a particular protein that you want to study, often you can get that made by particular cells, you can get cells to make that protein for you, but the preparation that you collect from the cells will be a mixture of misfolded proteins and correct proteins. There's a real challenge there from that mixture to pull out the protein in the correct shape that you want.

This technology demonstrated how you could take a difficult mixture of tangled up proteins and basically pull out the protein that you're interested in, and have it be refolded back into the correct shape.

ANDI HORVATH
Troy, you've got lots of students in your lab because it's a very active laboratory. What advice do you have for your students?

TROY ATTARD
A common question I get is, well it relates to what should I do in my career? I think my advice is actually talk to many people because everyone has different experiences. If you talk to me, I'll give you my experience, if you ask somebody else, they might have had a bad experience, they might have had a mean supervisor or whatever, and so that will colour the advice that they give. A student should broaden their horizons in terms of who they speak to.
That's getting advice, but if you're asking my advice, what I would tell them, I would say just follow what you're interested in, because I think getting into this field you have to take the opportunities that are presented to you.

I've got an example. One student who I'm mentoring from a couple of years ago, he's a very good student, he's got great marks, and he just wanted to have, or he wanted to get into a lab over summer just to get some bench top experience. It didn't really matter what he was doing. It was very difficult because he was up against other people or other students who were further advanced in their careers, so they had a great advantage over him.

Eventually he landed a position, I think it was for two months, to go to New South Wales and study Antarctic mosses, or Arctic mosses, and gain information on climate change based on these mosses that have been living for 500 years. He took that opportunity, now he's probably not going to end up being a researcher of mosses, but that would have given him a lot of experience with exposure in the field of research.

I think you've got to take your opportunities and then land in the area that interests you while you're slowly deciding what really interests you in the long term. That’s probably my advice to students, so don't stress out too much about not knowing exactly what you want to be, you just have to follow what interests you at the moment, and then as you get more exposure, you read more literature, you talk to more people, you'll get a better idea as time goes on what you really want to do.

ANDI HORVATH
Yes, there's a lot of happenstance involved, and luck, and sometimes you just don't know what leads to what, and doors that open that you couldn't have planned.

TROY ATTARD
That’s right, yes. Even if you talk to some of the professors here at Melbourne Uni and ask them what they did in their honours and their PhD, it'll probably be off left field compared to what they're doing now the more they think about research and where they fit into that. You can end up in a very different place, but I think it all starts with an interest in science and discovery, and you just take it from there.

ANDI HORVATH
Proteins and peptides are this invisible world to us, what would you like us to think about next time we hear the word peptides in the news?

TROY ATTARD
I think what we do as humans is, we think in pictures, for example you can't say don't think about an elephant, it pops into your head. I'm also a tutor of first year chemistry, and I often use imagery to help students imagine what might be going on with some of the chemistry that they learn about. I suppose using that approach you could think of peptides as segments of protein. If you think of protein like maybe a ball of twine, so it's a long linear string that is all scrunched up into a ball or various shapes. A peptide is just if you took a pair of scissors and snipped two little bits of a segment of that string, that would be your peptide.

I suppose in terms of the structure, if you're trying to imagine what might be going on on a molecular level, that's probably a good metaphor.

ANDI HORVATH
Dr. Troy Attard, thank you.

TROY ATTARD
Thanks a lot.

CHRIS HATZIS
Thank you to Dr Troy Attard, part-time application specialist in the Melbourne Protein Characterisation platform at the Bio21 Institute, University of Melbourne. And thanks to Dr Andi Horvath.

Eavesdrop on Experts - stories of inspiration and insights - was made possible by the University of Melbourne. This episode was recorded on September 29, 2020. You’ll find a full transcript on the Pursuit website. Production, audio engineering and editing by me, Chris Hatzis. Co-production - Silvi Vann-Wall and Dr Andi Horvath. Eavesdrop on Experts is licensed under Creative Commons, Copyright 2020, The University of Melbourne. If you enjoyed this episode, review us on Apple Podcasts and check out the rest of the Eavesdrop episodes in our archive. I’m Chris Hatzis. Join us again next time for another Eavesdrop on Experts.