Proteins are ubiquitous – they mediate almost all cellular processes, from signalling pathways to the transport of molecules and cell division. With funding provided by the DFG’s Emmy Noether programme, Dr. Oliver Schilling and his junior research group at the University of Freiburg, are developing tools that enable scientists to investigate the proteins of organisms in their functional context. Proteomics methods are helping Schilling’s team to understand a previously underestimated group of proteins – the proteases.
Proteases have for a long time been regarded as the body's waste cutters. However, these enzymes, which are able to cut the peptide bonds between amino acids, can do far more than just degrade defective proteins. Proteins play a key role in the proper functioning of signalling pathways. One example of such pathways is the coagulation cascade which begins immediately after a blood vessel has been damaged and brings the blood into contact with oxygen. Coagulation is triggered by proteases that activate coagulation enzymes by cutting off protein segments that would otherwise prevent the coagulation from taking place. "Over the last few years, it has become clear that proteases can be part of a broad range of signalling pathways," said Dr. Oliver Schilling, head of an Emmy Noether junior researcher group in Prof. Dr. Christoph Peters' department at the Institute of Molecular Medicine and Cell Research at the University of Freiburg. "But where in these networks do the proteases exert their action?"
There are about 550 different proteases in the human body. Several years ago, Dr. Thomas Reinheckel, one of Schilling's colleagues, drew attention to the importance of these molecules in the development of cancer. Reinheckel switched off the protease cathepsin B in mice suffering from cancer and found that the tumours started to grow more slowly and did not become as big as they would otherwise have become. As the molecules have huge medical potential, pharmaceutical companies are also interested in learning more about them. However, it has been shown in clinical studies that the application of protease inhibitors might be dangerous; unexpected side effects have occurred, but the molecular network of a specific protease is as yet unknown. "We can only fully discover the molecular network of a protease when we find the proteins that are altered by the proteases," said Schilling. "And proteomics methods have proven to be excellent tools for this task."
Proteomics refers to the large-scale study of all proteins (the proteome) of a biological system. It not only involves the study of the structure and sequence of molecules, but also their functional interactions. But how is it possible to find the target molecules that are cleaved and modified by the proteases? During his postdoctoral studies in Vancouver, Schilling and his then supervisor Professor Christopher Overall, developed methods that enabled them to screen biological material. The researchers investigated the proteins of cells lacking a specific protease and compared this protein pattern with cells that produced this specific protease. This enabled them to investigate the effect of a protease on the cellular proteome. Of equal importance for research are the target proteins and the cleavage sites of a protease. Since proteolysis (i.e. the process of cleaving proteins) generates new protein ends, the researchers developed methods that enabled them to have a detailed look at these ends and differentiate the "normal" protein ends from the cleavage sites introduced by the proteases under investigation.
The researchers use mass spectrometric and chromatographic methods to screen the proteome. They work in close cooperation with Dr. Martin Biniossek's team at the same institute. "Once we have determined the target proteins of a protease, the next steps will involve working with cell biologists in an attempt to find out what happens in a cell when the protease modifies its target proteins." The researchers at the Freiburg institute hope that this investigation will help them gain deeper insights into the biological function of cathepsin proteases.
Another major research priority of Schilling's group is the biochemical characterisation of proteases. The majority of proteases do not target specific protein sequences. Their active centres do not react with one particular protein, but can cut different proteins, even though the cleavage sites are not 100 percent identical. If the scientists are able to define the criteria a DNA sequence has to meet for a protease to be able to recognise it as target, this will also benefit applied research. For example, cell biologists will then be able to find out whether the cleavage products originate from the enzyme in question, or whether they are the products of a side reaction.
"The method we have developed enables us to find a large number of cleavage sequences," said Schilling. Up until now, about 8000 different target sequences have been known for 2000 known proteases. During his post-doctoral studies in the laboratory of Professor Overall, Schilling was able to detect about 2000 cleavage sequences for nine proteases. The method, known as ‘proteomic identification of protease cleavage sites - PICS', was published in 2008 in the renowned journal Nature Biotechnology. It is based on natural peptide libraries consisting of sequences of short protein fragments (peptides). The peptides were not synthesised chemically, but isolated from cells. The researchers incubate the peptide library with a protease that recognises peptides with matching target sequences and cleaves them. The researchers isolate the cleavage products from the peptide library and identify the individual peptides using mass spectrometry.
"Over the next few years, we will continue focusing on the development of new methods," said Schilling in the hope that he and his team will soon be able to identify the role of cathepsin proteases, for example, in molecular networks. This would represent a huge progress for both cancer research and the pharmaceutical industry.
Dr. Oliver Schilling Institute of Molecular Medicine and Cell ResearchUniversity of FreiburgTel: +49 761/203 9615 Fax: +49 761/203 9620 E-mail: oliver.schilling(at)mol-med.uni-freiburg.de