Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8.
Internally quenched fluorescent peptide substrates disclose the subsite preferences of human caspases 1, 3, 6, 7 and 8.
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DOI：
10.1042/bj3500563
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发表时间：
2000-09
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影响因子：
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通讯作者：
H. Stennicke;M. Renatus;M. Meldal;G. Salvesen
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文献类型：
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作者：
H. Stennicke;M. Renatus;M. Meldal;G. Salvesen
亚位点相互作用被认为定义了蛋白酶对其天然底物的严格特异性。为了探索导致细胞凋亡的蛋白水解途径中的这个问题，我们使用含有邻氨基苯甲酰基（也称为邻氨基苯甲酸）和3-硝基酪氨酸的内部淬灭的荧光肽底物，检测了人半胱天冬酶1、3、6、7和8的P（4）、P（1）和P（1）'亚位点偏好。以前的工作已经证明了S（4）亚位点在指导caspase家族特异性中的重要性。在这里，我们证明了S（1）和S（1）'亚位点的影响，侧翼的易断裂的肽键。半胱天冬酶的主要特异性决定位点S（1）亚位点表现出巨大的选择性，在该位点上对含有天冬氨酸的底物的切割优先性是谷氨酸的20000倍。因此，半胱天冬酶是已知的内肽酶中选择性最强的。我们发现半胱天冬酶在P（1）'位置显示出出乎意料的区分程度，一般偏好小的氨基酸残基，如丙氨酸、甘氨酸和丝氨酸，甘氨酸是优选的取代基。大的芳族残基也令人惊讶地良好耐受，但禁止带电荷的残基。虽然这描述了caspase家族中P（1）亚位点偏好的一般顺序，但在个体谱中存在一些差异，其中caspase-3特别混杂。总的来说，亚位点偏好可用于预测天然底物，但在某些情况下，通过寻找优选的肽切割位点不能预测推定的天然底物内的切割位点。在后一种情况下，我们得出结论，第二个网站的相互作用可能会克服，否则次优切割序列。
Subsite interactions are considered to define the stringent specificity of proteases for their natural substrates. To probe this issue in the proteolytic pathways leading to apoptosis we have examined the P(4), P(1) and P(1)' subsite preferences of human caspases 1, 3, 6, 7 and 8, using internally quenched fluorescent peptide substrates containing o-aminobenzoyl (also known as anthranilic acid) and 3-nitro-tyrosine. Previous work has demonstrated the importance of the S(4) subsite in directing specificity within the caspase family. Here we demonstrate the influence of the S(1) and S(1)' subsites that flank the scissile peptide bond. The S(1) subsite, the major specificity-determining site of the caspases, demonstrates tremendous selectivity, with a 20000-fold preference for cleaving substrates containing aspartic acid over glutamic acid at this position. Thus caspases are among the most selective of known endopeptidases. We find that the caspases show an unexpected degree of discrimination in the P(1)' position, with a general preference for small amino acid residues such as alanine, glycine and serine, with glycine being the preferred substituent. Large aromatic residues are also surprisingly well-tolerated, but charged residues are prohibited. While this describes the general order of P(1)' subsite preferences within the caspase family, there are some differences in individual profiles, with caspase-3 being particularly promiscuous. Overall, the subsite preferences can be used to predict natural substrates, but in certain cases the cleavage site within a presumed natural substrate cannot be predicted by looking for the preferred peptide cleavage sites. In the latter case we conclude that second-site interactions may overcome otherwise sub-optimal cleavage sequences.