Supramolecular Control of Enzyme Activity through Cucurbit[8]uril-Mediated Dimerization
Supramolecular Control of Enzyme Activity through Cucurbit[8]uril-Mediated Dimerization
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DOI：
10.1002/anie.201208239
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发表时间：
2013-01-01
影响因子：
16.6
通讯作者：
Brunsveld, Luc
中科院分区：
文献类型：
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作者：
Dang, Dung T.;Nguyen, Hoang D.;Brunsveld, Luc
蛋白质二聚化是一种普遍存在的调节蛋白质活性的机制，在广泛的生物学过程中，包括受体聚集，信号转导和凋亡。这些过程的分子控制对于阐明和干扰所涉及的蛋白质的分子机制至关重要。[1]为了服从小分子调节，感兴趣的蛋白质通常需要大量的蛋白质工程和引入结合小分子的另外的蛋白质结构域。[2-4]通过使用小的、遗传编码的肽基序来控制蛋白质二聚化在这方面是非常有吸引力的目标，因为短肽基序将对研究的蛋白质施加尽可能小的影响。然而，小分子通常不与短肽片段结合。合成的超分子分子，如甜甜圈形环糊精和葫芦脲[5]和其他更复杂的合成受体，[6，7]已被证明可以选择性地识别氨基酸和蛋白质元素。[8]葫芦脲是具有不同环尺寸的小凹主体分子，具有非常有前途的生物医学应用。[9-11]葫芦[7]脲可以选择性地识别胰岛素[12]或二茂铁修饰的蛋白质。[13葫芦[8]脲同时与两个短肽基序强而特异地结合。[15-17]分子结合机制涉及通过葫芦[8]脲腔内苯基的疏水相互作用和质子化N-末端胺官能团与葫芦[8]脲羰基边缘的相互作用选择性识别两个N-末端苯丙氨酸。[15]在荧光蛋白中引入N-末端苯丙氨酸-甘氨酸-甘氨酸（FGG）基序允许葫芦[8]脲在稀溶液中选择性地诱导这些蛋白质的同源二聚化。[17]因此，瓜环[8]具有作为功能性二聚化剂和蛋白质活化剂的巨大潜力。在本文中，描述了超分子蛋白质二聚化以控制酶活性的概念，使用葫芦[8]脲作为半胱天冬酶-9（casp-9）二聚化和活化的超分子诱导剂（图1a）。这种对蛋白质二聚化的超分子控制提供了一种独特的方法来可逆地控制半胱天冬酶二聚化和活化，这是仅使用经典蛋白质工程技术无法实现的。[18-21]在细胞中，casp-9主要以其非活性单体形式存在，仅在辅助因子诱导二聚化时才被激活。[22-25]例如，包括凋亡蛋白酶激活因子1和细胞色素c的核糖体负责募集
Protein dimerization is a ubiquitous mechanism to regulate protein activity in a broad range of biological processes including receptor clustering, signal transduction, and apoptosis. Molecular control over these processes is critical to elucidate and perturb the molecular mechanisms of the proteins involved.[1] To be amenable to small-molecule regulation, proteins of interest typically require substantial protein engineering and introduction of additional protein domains that bind the small molecule.[2–4] Control over protein dimerization by using a small, genetically encoded, peptide motif is a highly attractive target in this respect, as a short peptide motif would impose the smallest possible influence on the protein of study. Small molecules do not typically bind to short peptide fragments, however. Synthetic supramolecular molecules, such as the donut-shaped cyclodextrins and cucurbiturils [5] and other more complex synthetic receptors,[6, 7] have been shown to selectively recognize amino acids and protein elements.[8] Cucurbiturils are small concave host molecules of diverse ring sizes and feature highly promising biomedical applications.[9–11] Cucurbit [7] uril can selectively recognize insulin [12] or ferrocene-modified proteins.[13, 14] Cucurbit [8] uril strongly and specifically binds two short peptide motifs simultaneously.[15–17] The molecular binding mechanism involves the selective recognition of two N-terminal phenylalanines through hydrophobic interactions of the phenyl groups inside the cucurbit [8] uril cavity and interactions of the protonated N-terminal amine functionality with the carbonyl rim of the cucurbit [8] uril.[15] The introduction of an N-terminal phenylalanine-glycine-glycine (FGG) motif in fluorescent proteins allowed cucurbit [8] uril to selectively induce the homodimerization of these proteins in dilute solution.[17] Cucurbit [8] uril thus has great potential as a functional dimerizer and activator of proteins. Herein, the concept of supramolecular protein dimerization to control enzyme activity is described, using cucurbit [8] uril as a supramolecular inducer for caspase-9 (casp-9) dimerization and activation (Figure 1a). Such supramolecular control of protein dimerization provides a unique approach to reversibly control caspase dimerization and activation, not accessible by only using classical protein engineering techniques.Caspases are critical cysteine proteases in the apoptosis pathway and are responsible for cleaving proteins at specific aspartate residues.[18–21] In the cell, casp-9 exists primarily in its inactive monomeric form, becoming activated only upon induced dimerization by auxiliary factors.[22–25] For example, apoptosomes, which include the apoptotic protease activating factor 1, and cytochrome c are responsible for the recruitment