Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors
Bioassays to Monitor Taspase1 Function for the Identification of Pharmacogenetic Inhibitors
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DOI：
10.1371/journal.pone.0018253
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发表时间：
2011-05-25
期刊：
影响因子：
3.7
通讯作者：
Bier, Carolin
中科院分区：
文献类型：
--
作者：
Knauer, Shirley K.;Fetz, Verena;Bier, Carolin
背景：苏氨酸天冬氨酸酶1(Taspase1)介导混合系白血病(MLL)蛋白的切割，并引发MLL融合。与其他蛋白水解酶相比，对Taspase1的生物学相关性和功能的了解是有限的，因为目前还没有小分子抑制剂或基于细胞的功能分析方法。方法/研究结果：本文介绍了基于细胞的有效方法来探索Taspase1在体内的功能。它们由谷胱甘肽S转移酶、自身荧光蛋白变异体、Taspase1裂解位点和核进出口信号的合理组合组成。生物传感器主要定位于细胞质，而生物活性Taspase1的表达而不是非活性Taspase1突变体或蛋白酶Caspase3的表达触发了它们的蛋白水解性切割和核积累。与使用重组成分的体外检测相比，体内检测具有很高的效率。利用一种优化的核转位算法，该三色分析可适用于高通量显微镜平台(Z‘factor0.63)。使用自动化的高含量数据分析来筛选通过电子药效团筛选方法选择的重点化合物文库以及真菌提取物的集合。筛选鉴定出两个化合物，N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamide和2-苯基三氮唑-4，5-二羧酸，它们部分地抑制活细胞中Taspase1的切割。此外，该试验还被用来探测实体瘤细胞模型中的内源性Taspase1，并确定了有效切割Taspase1的改进的共识序列。这使得在电子计算机上识别新的假定的Taspase1靶标成为可能。这些基因包括含有FERM结构域的蛋白4B、酪氨酸蛋白磷酸酶Zeta和DNA聚合酶Zeta。结论：该方法不仅可以从基因水平上探讨Taspase1在体内的结构功能，也适用于高含量Taspase1抑制剂的筛选。这些工具将为Taspase1的S功能及其潜在的治疗意义提供新的见解。
Background: Threonine Aspartase 1 (Taspase1) mediates cleavage of the mixed lineage leukemia (MLL) protein and leukemia provoking MLL-fusions. In contrast to other proteases, the understanding of Taspase1's (patho) biological relevance and function is limited, since neither small molecule inhibitors nor cell based functional assays for Taspase1 are currently available.Methodology/Findings: Efficient cell-based assays to probe Taspase1 function in vivo are presented here. These are composed of glutathione S-transferase, autofluorescent protein variants, Taspase1 cleavage sites and rational combinations of nuclear import and export signals. The biosensors localize predominantly to the cytoplasm, whereas expression of biologically active Taspase1 but not of inactive Taspase1 mutants or of the protease Caspase3 triggers their proteolytic cleavage and nuclear accumulation. Compared to in vitro assays using recombinant components the in vivo assay was highly efficient. Employing an optimized nuclear translocation algorithm, the triple-color assay could be adapted to a high-throughput microscopy platform (Z'factor = 0.63). Automated high-content data analysis was used to screen a focused compound library, selected by an in silico pharmacophor screening approach, as well as a collection of fungal extracts. Screening identified two compounds, N-[2-[(4-amino-6-oxo-3H-pyrimidin-2-yl)sulfanyl]ethyl]benzenesulfonamide and 2-benzyltriazole-4,5-dicarboxylic acid, which partially inhibited Taspase1 cleavage in living cells. Additionally, the assay was exploited to probe endogenous Taspase1 in solid tumor cell models and to identify an improved consensus sequence for efficient Taspase1 cleavage. This allowed the in silico identification of novel putative Taspase1 targets. Those include the FERM Domain-Containing Protein 4B, the Tyrosine-Protein Phosphatase Zeta, and DNA Polymerase Zeta. Cleavage site recognition and proteolytic processing of these substrates were verified in the context of the biosensor.Conclusions: The assay not only allows to genetically probe Taspase1 structure function in vivo, but is also applicable for high-content screening to identify Taspase1 inhibitors. Such tools will provide novel insights into Taspase1's function and its potential therapeutic relevance.