Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy
Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy
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DOI：
10.1039/c2mb25144b
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发表时间：
2012-01-01
影响因子：
--
通讯作者：
Sieber, Stephan A.
中科院分区：
文献类型：
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作者：
Eirich, Juergen;Burkhart, Jens L.;Sieber, Stephan A.
微管（mt）是由α -和β -微管蛋白单体组成的高度动态聚合物，存在于所有分裂和非分裂细胞中。已知存在各种各样的天然产物，通过稳定或破坏这些绳状聚合物来干扰微管网络。其中，管溶蛋白代表了一类新的和有效的细胞抑制四肽起源于粘杆菌。早期研究表明，微管溶素通过抑制微管蛋白聚合与真核细胞骨架相互作用，EC50值在皮摩尔范围内。最近，预管溶蛋白被描述为保持其更复杂的管溶蛋白亲戚的高管蛋白降解活性，并且代表了一种更容易合成的有效合成靶标。虽然微管蛋白被认为是微管蛋白的专用靶点，但到目前为止还没有在整个蛋白质组的背景下对前微管蛋白进行全面的分子靶点分析。在这里，我们利用合成化学开发了两种前管溶素光亲和探针，用于基于细胞活性的蛋白质分析和成像研究，以揭示和可视化专用靶点。我们的研究结果清楚地表明，pre - tubulysin对β -微管蛋白具有显著的选择性，我们通过基于质谱的蛋白质组学分析平台以及基于微管蛋白抗体的完整细胞共染色独立证实了这一点。
Microtubules (mt) are highly dynamic polymers composed of alpha- and beta-tubulin monomers that are present in all dividing and non-dividing cells. A broad variety of natural products exists that are known to interfere with the microtubule network, by either stabilizing or de-stabilizing these rope-like polymers. Among those tubulysins represent a new and potent class of cytostatic tetrapeptides originating from myxobacteria. Early studies suggested that tubulysins interact with the eukaryotic cytoskeleton by inhibition of tubulin polymerization with EC50 values in the picomolar range. Recently, pretubulysins have been described to retain the high tubulin-degradation activity of their more complex tubulysin relatives and represent an easier synthetic target with an efficient synthesis already in place. Although tubulin has been suggested as the dedicated target of tubulysin a comprehensive molecular target analysis of pretubulysin in the context of the whole proteome has not been carried out so far. Here we utilize synthetic chemistry to develop two pretubulysin photoaffinity probes which were applied in cellular activity-based protein profiling and imaging studies in order to unravel and visualize dedicated targets. Our results clearly show a remarkable selectivity of pretubulysin for beta-tubulin which we independently confirmed by a mass-spectrometry based proteomic profiling platform as well as by tubulin antibody based co-staining on intact cells.