Fluorescent Peptomer Substrates for Differential Degradation by Metalloproteases
Fluorescent Peptomer Substrates for Differential Degradation by Metalloproteases
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DOI：
10.1021/acs.biomac.2c01077
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发表时间：
2022-10-21
影响因子：
6.2
通讯作者：
Rosales，Adrianne M.
中科院分区：
文献类型：
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作者：
Austin，Mariah J.;Schunk，Hattie;Rosales，Adrianne M.
蛋白水解酶，尤其是MMPs，由于其在生理和病理过程中的核心作用，是极具吸引力的生物标志物。然而，由于底物特异性图谱的重叠，区分可降解底物的基质金属蛋白酶活性是一项困难的任务。在这里，我们开发了一个肽聚体系统(肽-类肽杂交体)来探索非天然残基对基质金属蛋白酶特异性的影响。类肽是一种非天然的N-取代的甘氨酸，具有很大的侧链多样性。鉴于在MMPs共有序列的P3位存在一个标志性的Pro残基，我们假设类肽可能提供了N-取代的替代品来产生与MMPs的差异相互作用。为了验证这一假设，将胃肠底物与5种不同的MMPs和细菌胶原酶接触，并通过荧光共振能量转移和液质联用分别监测裂解速率和降解片段的组成。我们发现，在P3和P3‘底物部位对类肽残基有很好的耐受性，这些部位类肽的同一性对切割速度有适度的影响。然而，在活性与侧链同一性的相关性比侧链位置更强的P1底物位置上，类肽残基的耐受性更好。所研究的所有MMPs对消化酶的特异性表现出相似的趋势，但在蛋白分解速率上表现出不同程度的差异。这些动力学图谱作为这些酶的“指纹”，并通过多变量数据分析得到分离。为了进一步证明这种可调性在降解动力学中的实际应用，将消化酶底物连接到水凝胶中，并在不同的时间尺度上释放。总体而言，这项工作代表着朝着最大化不同的基质金属蛋白酶行为的探针设计迈出了重要的一步，并提出了利用类肽取代调整降解动力学的设计规则，这对使用基于阵列的传感器的诊断和预后应用具有潜在的意义。
Proteases, especially MMPs, are attractive biomarkers given their central role in both physiological and pathological processes. Distinguishing MMP activity with degradable substrates, however, is a difficult task due to overlapping substrate specificity profiles. Here, we developed a system of peptomers (peptide–peptoid hybrids) to probe the impact of non-natural residues on MMP specificity for an MMP peptide consensus sequence. Peptoids are non-natural,N-substituted glycines with a large side-chain diversity. Given the presence of a hallmark proline residue in the P3 position of MMP consensus sequences, we hypothesized that peptoids may offerN-substituted alternatives to generate differential interactions with MMPs. To investigate this hypothesis, peptomer substrates were exposed to five different MMPs, as well as bacterial collagenase, and monitored by fluorescence resonance energy transfer and liquid chromatography–mass spectrometry to determine the rate of cleavage and the composition of degraded fragments, respectively. We found that peptoid residues are well tolerated in the P3 and P3′ substrate sites and that the identity of the peptoid in these sites displays a moderate influence on the rate of cleavage. However, peptoid residues were even better tolerated in the P1 substrate site where activity was more strongly correlated with side-chain identity than side-chain position. All MMPs explored demonstrated similar trends in specificity for the peptomers but exhibited different degrees of variability in proteolytic rate. These kinetic profiles served as “fingerprints” for the proteases and yielded separation by multivariate data analysis. To further demonstrate the practical application of this tunability in degradation kinetics, peptomer substrates were tethered into hydrogels and released over distinct timescales. Overall, this work represents a significant step toward the design of probes that maximize differential MMP behavior and presents design rules to tune degradation kinetics with peptoid substitutions, which has promising implications for diagnostic and prognostic applications using array-based sensors.