Please could you tell us a little bit about rhomboid enzymes? Where are they found and what do they do?
Rhomboid enzymes live in the cell’s membrane where their job is to cut the anchors of proteins, which releases the target proteins from the membrane. This could be because the released protein is needed elsewhere, or it might need to be removed from the membrane because it’s damaged.
What makes rhomboid enzymes so fascinating is that they’re present in almost all cells on Earth, from bacteria straight to humans. This implies that they do something that’s really useful for cells.
What was previously known about rhomboid enzymes and what did your research discover?
The central mystery in biochemical terms is how this enzyme can function within the membrane, because that’s not a place where such enzymes were thought to be able to work.
Five years ago several labs succeeded in solving the structure of a bacterial rhomboid enzyme. That was a major breakthrough, and it meant that we could see almost every atom in the molecule.
However, by just looking at its architecture, it was hard to understand what each part did. It’s like looking at a house – you can see all walls, but you don’t know which ones keep it up and which can be moved around to make different rooms. We set out to figure out what each part of rhomboid does to contribute to the enzyme’s architecture versus its function.
How did you make these discoveries?
The key was to figure out a way to measure rhomboid enzyme stability independently from its enzyme function. This is particularly tricky with proteins that live in the membrane. In fact, decades of work by labs all over the world has resulted in such measurements being achieved for only a handful of membrane enzymes.
We turned to a new technology that heats samples and measures light that’s bouncing off the enzymes. As more heat is applied, the enzyme eventually ‘breaks’ and that’s detected as a spike in light. We could then change individual parts of rhomboid enzymes and determine which caused the altered enzymes to break with less heat applied.
We ultimately made and purified over 150 such rhomboid variants and determined their breaking point and enzyme function. This allowed us to map precisely what parts contribute to rhomboid stability and how – we now have essentially a full architectural ‘heat-map’ of the entire enzyme, and that’s really exciting. We now see the enzyme structure in a whole new light!
It was noted in the news that some enzymes lost their function despite there being no obvious change in their shape or stability. Do you have any ideas for why this occurred?
We hope to learn a lot from these rare variants – there are few things more informative in science than those experiments that give us the ‘I didn’t expect that at all’ result. So far we have studied only one such class and found that they are important for keeping water inside the enzyme.
Rhomboid enzymes use water to cut their target protein anchors, but the membrane is a place in the cell that’s devoid of water. A few of the parts of the enzyme that, when altered, gave the same enzyme stability but the enzyme no longer function are involved in bringing water to the part of the enzyme where it’s needed. Again, we didn’t anticipate this at the start, and were really fortunate to be working closely with a great research group at NYU that was doing computer simulations of how the enzyme moves. Together, our results meshed into a brand new idea – a ‘water-retention site’ inside the enzyme. Even for enzymes, it’s important to stay hydrated!
What implications does your research have for the treatment of malaria?
The long-term application of our research is to design drugs that block rhomboid enzyme action in malaria and other parasites. From our previous research we believe that rhomboid function is essential for parasites to infect human cells. So in simple terms, no rhomboid enzyme function, no parasite infection. But to achieve this goal you need to know how an enzyme’s put together in the first place.
Our new, comprehensive heat-map of rhomboid’s architectural features takes us one step closer to that eventual goal, because enzymes that are rock-solid behave differently in terms of drug design compared to those that are ‘squishy’.
Do you think your research will have any implications for the treatment of other diseases? What are your plans for further research into this field?
Yes; this could be the most exciting part. Over the past few years it’s become clear that rhomboid enzymes participate in the infective cycles of many diverse parasites. These range from Toxoplasma that infects hundreds of thousands in the US, to amoebae that cause millions of cases of dysentery worldwide each year.
So it’s conceivable that anti-rhomboid drugs could be used to treat a whole spectrum of very different diseases. But that’s far off into the future and what’s needed as the next step is more research on the parasite rhomboid enzymes directly, because some of these enzymes are 3 times larger than the bacterial enzymes that we studied. The enzyme core is recognizably similar, but we don’t know what these other parts do, so we need to study them directly.
How do you think the future of our knowledge of rhomboid enzymes will progress?
It’s been just over a decade now since I discovered the enzymatic function of rhomboid. In that time, the field has moved by leaps and bounds: we now have an atomic map of the molecule and some really sophisticated methods to study how they work, and an ever-growing list of important functions in cell biology and disease.
I think that we’re now going to start seeing all of these advances start coming together, and hopefully allow us to think about designing therapeutic strategies on a realistic level. There’s a lot of work ahead, but the needed tools are coming online fast so the future is likely to be bright. I, for one, am really excited.
How do you think the future of malaria treatment will progress?
Sequencing of the malaria parasite genome a decade ago and the application of cutting-edge molecular and genetic technologies have really started to unravel the deepest, darkest secrets of how these deadly parasites achieve their sinister goals. Parasitologists are discovering many unexpected aspects of the bug’s unusual cell biology and metabolism that could be very effective and specific targets for therapeutic intervention.
There are so many ingenious ideas out there right now, from blocking unusual parasite pathways, to novel enzyme targets like rhomboid, to infecting mosquitoes with agents that kill malaria parasites while they’re still in the mosquitoes themselves far before they get near a human.
Part of the accelerating pace of discovery has been fuelled by bold funding initiatives for basic research. Here at Hopkins, a philanthropic gift from Mike Bloomberg has allowed people to come up with wild, fresh ideas and run with them. It will be fascinating to see these bold initiatives transformed into the medicines of tomorrow.
Where can readers find more information?
About Associate Professor Sinisa Urban
Sin Urban is an Associate Professor at the Johns Hopkins University School of Medicine (USA) and the Howard Hughes Medical Institute (HHMI). He received his B.Sc. in Honours Genetics at the University of Alberta (Canada), where he studied viral replication. He obtained his Ph.D. in 2002 from the University of Cambridge (UK), where he discovered that the signaling factor rhomboid is an intramembrane protease.
Dr Urban continued his research in Cambridge as a JB & Millicent Kaye Prize Fellow in Cancer Studies, and then in 2004 started his independent lab as a Harvard Fellow at Harvard Medical School.
Dr Urban was recruited to Johns Hopkins in 2006, where his research now encompasses investigating the pathogenic functions and biochemical mechanisms of intramembrane proteases. Dr Urban is also Director of Admissions of the Biochemistry, Cellular and Molecular Biology PhD program at Johns Hopkins, a fellow of the Packard Foundation, and serves on the Governing Council of the International Proteolysis Society and the Editorial Board of the Journal of Biological Chemistry.
Dr Urban has received numerous awards for his work, including: the Genetics Society of America Sandler Award for Outstanding Ph.D. Dissertation, Oxford’s Rolleston and MRC’s Max Perutz Prizes for Original Research, HRH the Princess of Wales’ Canada Scholarship for AIDS Research, the Cambridge Gedge Prize for Outstanding Observations in Physiology, and a career award from the Burroughs-Wellcome Fund.
Malaria crystal structure offers clues for more effective medications