STUDIES OF THE FUNCTION

The High Temperature Requirement A (HtrA) family of proteins plays an important role in protein quality control, and loss of function is directly associated with several serious diseases. We spoke to Dr Björn Burmann about his research into how these proteases work on the molecular level, which could lay the foundations for further exploitations of these proteases in the future.

The HtrA family of proteins are thought to play a significant role in the development and progression of several different diseases. Experimental evidence suggests that loss of proteolytic activity is a key factor in certain types of cancer, as well as some neurodegenerative conditions. As an Assistant Professor at the Wallenberg Centre for Molecular and Translational Medicine, Dr Björn Burmann aims to dive deeper into the structure and function of these proteins. “In my lab we are looking in detail into how they actually work on the molecular level,” he outlines. There are four of these proteins in humans within the HtrA (high temperature requirement A) family, and they function as proteases, helping to break down or cleave proteins and aggregates. “One of the main functions of proteases is to kind of chop down proteins, to degrade them,” explains Dr Burmann.

HtrA proteases There is still much to learn about these HtrA proteases however, in particular how their function is modulated by interactions with aggregated proteins, a topic central to Dr Burmann’s research. One part of this work involves using nuclear magnetic resonance (NMR) spectroscopy in solution to gain deeper insights into these proteases. “We do a lot of biochemistry and use other biophysical analysis techniques to study protein interactions and their consequences. In order to get information on the atomic level about these proteins as well as structural and dynamical changes, we need to use NMR,” outlines Dr Burmann. This is a structural biology technique allowing Dr Burmann and his colleagues to analyse proteases in great depth. “We can look at the proximity of amino-acids in a 3-dimensional structure for example. With NMR we get a picture of how a protein behaves in solution,” he says.

A number of other techniques are also being utilised in research, including X-ray crystallography and cryo-electron microscopy. These techniques are highly complementary to NMR spectroscopy, says Dr Burmann. “We’re making use of these different techniques, combining them in the most convenient way possible to answer biological questions,” he outlines. A lot of attention in Dr Burmann’s group is focused on the HtrA2 protease in particular, which is involved in triggering apoptosis, the controlled cell death program. “HtrA2 resides in the membrane of mitochondria. The signal which leads to the initial cleavage and which effectively activates this protein is unknown,” he explains. “On a cartoon level, it is known that HtrA2 has to be effectively detached from this membrane through protein cleavage.”

Preparing E. coli cultures to express recombinant HtrA2.

Putting an HtrA2-sample into an NMR spectrometer.

This apoptosis function needs to be tightly regulated, as dysfunction can eventually lead to serious health problems. However, the underlying mechanisms behind how HtrA2 is regulated in the body are not yet well understood. “First there is protein cleavage, then HtrA2 is further activated by phosphorylation. But we’re missing a lot of details about how this activation actually happens and the individual steps involved,” says Dr Burmann. One possibility is that there is a kind of stress signal in mitochondria, which then triggers further events. “It looks like it’s a multi-step process, but our knowledge is fairly limited at this point. There might also be alternative activation mechanisms,” continues Dr Burmann.

Researchers are also looking at how the HtrA2 protein interacts with several known partners, including the α-synuclein protein, which is associated with Parkinson’s disease. A deeper understanding of the structure and function of these proteins could help lay the foundations for future drug development, believes Dr Burmann. “We hope to contribute important new information, which down the road might lead to the identification of small molecule effectors,” he explains. However, the main focus in research at this stage is on understanding the fundamental behaviour of these proteins. “We are now starting to look into how it recognises its substrates, and how it does the job of proteolytic cleavage,” continues Dr Burmann.

With solution NMR we get a detailed picture of how the protein behaves in solution. We are now starting to look into how it recognises its substrates, and how it does the job of proteolytic cleavage.

By using solution NMR and other structural biology techniques to study proteases, together with biophysical methods, Dr Burmann and his colleagues hope to shed new light in this area. NMR provides very rich information about structure and dynamical changes in solutions, from which researchers can then look to gain deeper functional insights. “We can put the protein in a test tube and study it in a timedependent manner, over different timescales. Then we can look at things like how does phosphorylation occur? Over what time period does phosphorylation occur in vitro?” he outlines. Phosphorylation is a form of chemical modification of the protein, where a phosphor group is added to the protein. “We are trying to understand what phosphorylation does to the behaviour or functionality of HtrA,” explains Dr Burmann.

The next step beyond this point will be to develop an experimentally-driven model of how the protein works on the atomic level. This would potentially enable researchers to predict how the protein would react to the presence of different substrates, invaluable information in terms of future drug development. “We could offer suggestions, so that we understand what that protein does upon recognising the substrate, and how it then goes on to cleave it. What parts of the protein are important for this?” outlines Dr Burmann. Once researchers understand how these proteins work, they can then look towards manipulating them in certain ways to improve therapeutic outcomes. “The longterm goal would be to understand how these proteins work and what goes wrong when they don’t function properly,” says Dr Burmann.

STUDIES OF THE FUNCTION Studies of the function of HtrA protease at atomic level by NMR spectroscope Project Objectives The overall aim of the study is the determination of the structural and dynamical properties of the oligomeric human mitochondrial HtrA2–protease, its structural adaptions upon maturation, the structural basis of substrate recognition, and elucidating its role in apoptosis and neurodegenerative diseases. Project Funding This project is funded by the Swedish Research Council (Vetenskaprådet 2016-04721). Contact Details Project Coordinator, Dr. Björn Burmann, Assistant Professor Department of Chemistry and Molecular Biology Wallenberg Centre for Molecular and Translational Medicine University of Gothenburg P.O. Box 462 SE-405 30 Gothenburg T: +46 31 786 3937 E: bjorn.marcus.burmann@gu.se W: www.wcmtm.gu.se/research-groups/burmann

The Swedish NMR Centre of the University of Gothenburg is acknowledged for spectrometer time.

Dr Björn Burmann

Dr Björn Burmann is Associate Senior Lecturer in Chemistry at the Wallenberg Centre for Molecular and Translational Medicine, part of the University of Gothenburg. He investigates macromolecular protein machines which underly essential cellular functions using high resolution NMR. He aims to elucidate the function of these machines at the atomic level, in order to understand their dysfunction in certain diseases.