A small molecule with differential effects on the PTS1 and PTS2 peroxisome matrix import pathways
A small molecule with differential effects on the PTS1 and PTS2 peroxisome matrix import pathways
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DOI：
10.1111/j.1365-313x.2010.04473.x
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发表时间：
2011-03-01
期刊：
影响因子：
7.2
通讯作者：
Baker, Alison
中科院分区：
文献类型：
--
作者：
Brown, Laura-Anne;O'Leary-Steele, Catherine;Baker, Alison
小分子的使用在剖析生物过程方面具有巨大的力量。本研究提出了过氧化物酶体基质蛋白输入抑制剂的鉴定和表征。进行微型筛选以鉴定引起过氧化物酶体形态改变或过氧化物酶体靶向荧光报告蛋白的错误定位的分子。鉴定了一种苯并咪唑先导化合物（LDS-003655），其导致过氧化物酶体中GFP荧光降低和细胞溶质GFP蓄积。该化合物的作用对过氧化物酶体是特异性的，因为即使在25 μ M时，高尔基体、内质网和肌动蛋白细胞骨架也不受影响，而在100 nM时，通过PTS 1途径的过氧化物酶体输入会受到损害。当幼苗在25 μ M LDS-003655上生长时，它们显示出在生长素存在下生长的幼苗的典型形态，并且诱导了生长素报告基因DR 5：：GFP的表达。与已知的生长素相比，LDS-003655衍生物的集中文库的分析得出以下结论：LDS-003655的生长素样活性归因于其原位水解产生2，5-二氯苯甲酸，而LDS-003655的输入抑制活性需要整个分子。没有一种生长素对过氧化物酶体蛋白的输入有任何影响。通过PTS 2输入途径的基质输入对LDS-003655及其活性类似物相对不敏感，仅在高浓度下长时间孵育后观察到影响。在存在100 nM LDS-003655的情况下，PTS 1输入途径受体PEX 5的稳态蛋白水平降低，表明输入抑制的可能机制。
The use of small molecules has great power to dissect biological processes. This study presents the identification and characterisation of an inhibitor of peroxisome matrix protein import. A mini-screen was carried out to identify molecules that cause alteration in peroxisome morphology, or mislocalization of a peroxisome targeted fluorescent reporter protein. A benzimidazole lead compound (LDS-003655) was identified that resulted in reduced GFP fluorescence in peroxisomes and cytosolic GFP accumulation. The effect of the compound was specific to peroxisomes as Golgi bodies, endoplasmic reticulum and the actin cytoskeleton were unaffected even at 25 mu M, whereas peroxisome import via the PTS1 pathway was compromised at 100 nM. When seedlings were grown on 25 mu M LDS-003655 they displayed morphology typical of seedlings grown in the presence of auxin, and expression of the auxin reporter DR5::GFP was induced. Analysis of a focussed library of LDS-003655 derivatives in comparison with known auxins led to the conclusion that the auxin-like activity of LDS-003655 is attributable to its in situ hydrolysis giving rise to 2,5-dichlorobenzoic acid, whereas the import inhibiting activity of LDS-003655 requires the whole molecule. None of the auxins tested had any effect on peroxisome protein import. Matrix import by the PTS2 import pathway was relatively insensitive to LDS-003655 and its active analogues, with effects only seen after prolonged incubation on high concentrations. Steady-state protein levels of PEX5, the PTS1 import pathway receptor, were reduced in the presence of 100 nM LDS-003655, suggesting a possible mechanism for the import inhibition.