New approaches to handle old compounds – Studying the ecological function and bioactivity of Microcystins exploiting bio-orthogonal chemistry

微囊藻毒素（Microcystins，MCs）是由不同的蓝藻属（如微囊藻属（Microcystis））产生的环状七肽。作为淡水蓝藻水华产生的主要毒素，它们对环境产生重大影响，有时甚至威胁动物或人类的生命。由于它们的重要性，MC属于最好的研究蓝藻毒素。然而，MC的生物学作用的生产生物体，以及它们的多因素的真核细胞的作用模式仍然没有完全理解。该项目的目标是有助于了解这些重要的蓝藻毒素的生物学功能和作用模式。为了达到这一目标，我们将致力于回答以下问题：在微囊藻的MC生产细胞中，哪些蛋白质与MC结合？MCs位于生产细胞的何处？除了目前已知的靶点外，MCs被真核细胞摄取后还与哪些蛋白质结合？我们将使用我们新开发的“可点击”MC来解决这些问题。这些化合物在微囊藻中原位产生，具有可用于生物正交交联技术的反应性官能团，即所谓的“点击反应”。我们可以将这些MC共价结合到标记物上，如荧光染料或金纳米颗粒，也可以将它们附着到聚合物上，如功能化的琼脂糖珠。将在这个项目中使用的主要技术是下拉蛋白质组学，以确定哪些蛋白质MC结合，和电子显微镜分析MC在生产细胞中的定位。这些化合物也可用于“目标捕捞”，以识别真核细胞中的结合伴侣，并用于荧光显微镜研究MC在细胞中的摄取和分布。这将使我们能够比以前的研究更详细地研究MC的生物学作用。

Microcystins (MCs) are cyclic heptapeptides produced by different cyanobacterial genera, e.g. Microcystis. As major toxins produced during freshwater cyanobacterial blooms, they have a significant impact on the environment, and sometimes even threaten animal or human life. Due to their importance, MCs belong to the best studied cyanobacterial toxins. However, the biological role of MCs for the producing organisms as well as their multifactorial mode of action on eukaryotic cells are still not fully understood. The goal of this project is to contribute to the understanding of the biological functions and the mode of action of these important cyanotoxins. To reach this goal, we will work on answering the following questions: To which proteins bind MCs in MC producing cells of Microcystis? Where are MCs localized in the producing cells? To which proteins bind MCs after uptake into the eukaryotic cell besides the targets known to date?We will address these questions using our newly developed “clickable” MCs. These compounds, produced in situ in Microcystis, feature reactive functional groups that can be used for bio-orthogonal crosslinking techniques, so-called “click reactions“. We can covalently bind these MCs to labels such as fluorescent dyes or gold nanoparticles, and also attach them to polymers such as functionalized agarose beads. The major techniques that will be used in this project are pull-down proteomics to identify to which proteins MCs do bind, and electron microscopy to analyze the localization of MCs in the producing cells. The compounds can also be used for “target fishing” to identify binding partners in eukaryotic cells and for fluorescence microscopy to study MC uptake and distribution in cells. This will enable us to study the biological roles of MCs in greater detail than it has been possible in previous studies.