Heparan sulfate - a master regulator of matrix metalloproteinase biology?

信号蛋白如形态因子、细胞因子或趋化因子的细胞外切割是一种不可逆的翻译后修饰，对细胞的细胞外信号传导过程至关重要。为了更好地表征这些调控过程并了解其相关的病理情况，新的基于质谱（MS）的蛋白质组学方法已经开发出来，可以将底物加工与体外和体内的蛋白酶联系起来。然而，迄今为止，蛋白酶筛选通常是人工的，并且不能解释蛋白酶如何在体内以特定的和生理相关的方式切割其底物。这种差异的一个原因可能是细胞外基质（ECM）不仅作为许多蛋白酶的底物，如基质金属蛋白酶（MMP）家族的成员，而且还通过结合许多底物和蛋白酶在调节细胞外蛋白水解中发挥积极作用。硫酸肝素（HS）蛋白聚糖（HSPGs）是ECM的重要组成部分，通过其可变硫酸盐化的HS侧链结合多种形态因子、细胞因子和趋化因子，从而调节其生物活性。这提出了HS通过结合和共定位相关分子来调节蛋白酶底物特异性的可能性，以及HS直接调节底物裂解位点的可及性或蛋白酶活性的可能性。在我的博士论文中，我已经可以展示HS在形态原Hedgehog的切割和释放中的调节作用。现在，我的目标是通过比较具有不同HS表达的脊椎动物细胞系的分泌组，将HS描述为细胞外蛋白水解的主要调节因子，该技术是由丹麦技术大学Ulrich auf dem Keller教授的实验室开发和应用的最先进的MS蛋白质组学技术。许多MMPs特异性结合HS，因此这些细胞系也将用于确定HS对炎症相关分泌蛋白酶MMP9的活性和特异性、对其底物的稳定性和差异加工以及蛋白酶水平的影响。最后，在不同HS变异体存在的情况下，选择趋化因子候选物来研究HS调节的MMP9切割。hs依赖的底物切割将通过已知的趋化因子活性测定和靶向蛋白质组学进行生化验证。所获得的数据不仅将使ms蛋白酶筛选更具生理学相关性，而且还将在全球范围内为基质调节的细胞外蛋白水解提供新的见解。

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.