Heparan sulfate - a master regulator of matrix metalloproteinase biology?

点击翻译按钮获取中文摘要

Extracellular cleavage of signaling proteins such as morphogens, cytokines or chemokines, an irreversible post-translational modification, is crucial for extracellular signaling processes of cells. In order to better characterize such regulatory processes and understand their associated pathological situations, novel mass spectrometry (MS)-based proteomics methods have been developed, which allowed linking substrate processing to proteases in vitro and in vivo. However, to date, protease screenings are often artificial and do not explain how proteases cleave their substrates in a specific and physiologically relevant manner in vivo. One reason for this discrepancy may be that the extracellular matrix (ECM) not only serves as a substrate for many proteases, like members of the matrix metalloproteinase (MMP) family, but also plays an active role in regulating extracellular proteolysis by binding many substrates and proteases. Heparan sulfate (HS) proteoglycans (HSPGs), which are important ECM components, bind many morphogens, cytokines, and chemokines via their variably sulfated HS side chains and thereby regulate their biological activity. This raises the possibilities that HS regulates protease substrate specificity by binding to and co-localizing the molecules involved and that HS directly modulates substrate cleavage site accessibility or protease activity. During my PhD thesis I could already show such a regulatory role of HS in the cleavage and release of the morphogen Hedgehog. Now, I aim to characterize HS as one main regulator of extracellular proteolysis by comparing the secretomes of vertebrate cell lines with different HS expression through novel state-of-the-art MS proteomics technologies, developed and applied in the laboratory of Prof. Dr. Ulrich auf dem Keller at the Technical University of Denmark. Many MMPs specifically bind to HS, thus these cell lines will also be used to determine the effect of HS on the activity and specificity of the inflammation related secreted protease MMP9, on the stability and differential processing of its substrates, and on the protease level. Lastly, HS-modulated MMP9 cleavage will be studied with selected chemokine candidates in the presence of different HS variants. HS-dependent substrate cleavage will be verified biochemically, with known chemokine activity assays and targeted proteomics. The data obtained will not only make MS-protease screenings more physiological relevant, but also provide novel insights into matrix regulated extracellular proteolysis on a global scale.