<br>与半胱氨酸组织蛋白酶相比，组织蛋白酶 B 具有独特的切割特性，可在较宽的 pH 范围内设计和验证组织蛋白酶 B 的特定底物,Biochemistry

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与半胱氨酸组织蛋白酶相比，组织蛋白酶 B 具有独特的切割特性，可在较宽的 pH 范围内设计和验证组织蛋白酶 B 的特定底物
Biochemistry ( IF 2.9 ) Pub Date : 2023-07-17 , DOI: 10.1021/acs.biochem.3c00139
Michael C Yoon _{1,

2} , Von Phan _{1,

2} , Sonia Podvin ₁ , Charles Mosier ₁ , Anthony J O'Donoghue ₁ , Vivian Hook _{1,

2,

3}

Affiliation

组织蛋白酶 B 的生物学和病理功能发生在酸性溶酶体以及细胞质、细胞核和细胞外位置的中性 pH 值下。重要的是，与中性 pH 条件相比，组织蛋白酶 B 在酸性 pH 条件下表现出不同的底物裂解特性。因此，需要开发组织蛋白酶 B 的特定底物来测量其在广泛 pH 范围内的活性。目前用于监测组织蛋白酶 B 活性的底物由 Z-Phe-Arg-AMC 和 Z-Arg-Arg-AMC 组成，但它们缺乏特异性，因为它们被其他半胱氨酸组织蛋白酶切割。此外，Z-Arg-Arg-AMC 可监测中性 pH 下的组织蛋白酶 B 活性，并在酸性 pH 下显示出最低的活性。因此，本研究的目的是设计和验证特定的荧光肽底物，这些底物可以在从酸性到中性 pH 条件的广泛 pH 范围内监测组织蛋白酶 B 的活性。通过质谱法在 pH 4.6 和 pH 7.2 下进行多重底物分析，将组织蛋白酶 B 与半胱氨酸组织蛋白酶 K、L、S、V 和 X 的深度裂解特性进行比较。切割偏好分析预测三肽 Z-Nle-Lys-Arg-AMC 作为组织蛋白酶 B 的首选底物。值得注意的是，Z-Nle-Lys-Arg-AMC 表现出测量高组织蛋白酶 B 比活性的有利特性。中性 pH 值，并且比其他半胱氨酸组织蛋白酶更容易被组织蛋白酶 B 特异性切割。 Z-Nle-Lys-Arg-AMC 专门监测神经元和神经胶质细胞中的组织蛋白酶 B 活性，这与组织蛋白酶 B 蛋白的相对丰度一致。这些发现验证了 Z-Nle-Lys-Arg-AMC 作为一种新型底物，可以在广泛的 pH 范围内专门监测组织蛋白酶 B 的活性。

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Distinct Cleavage Properties of Cathepsin B Compared to Cysteine Cathepsins Enable the Design and Validation of a Specific Substrate for Cathepsin B over a Broad pH Range

The biological and pathological functions of cathepsin B occur in acidic lysosomes and at the neutral pH of cytosol, nuclei, and extracellular locations. Importantly, cathepsin B displays different substrate cleavage properties at acidic pH compared to neutral pH conditions. It is, therefore, desirable to develop specific substrates for cathepsin B that measure its activity over broad pH ranges. Current substrates used to monitor cathepsin B activity consist of Z-Phe-Arg-AMC and Z-Arg-Arg-AMC, but they lack specificity since they are cleaved by other cysteine cathepsins. Furthermore, Z-Arg-Arg-AMC monitors cathepsin B activity at neutral pH and displays minimal activity at acidic pH. Therefore, the purpose of this study was to design and validate specific fluorogenic peptide substrates that can monitor cathepsin B activity over a broad pH range from acidic to neutral pH conditions. In-depth cleavage properties of cathepsin B were compared to those of the cysteine cathepsins K, L, S, V, and X via multiplex substrate profiling by mass spectrometry at pH 4.6 and pH 7.2. Analysis of the cleavage preferences predicted the tripeptide Z-Nle-Lys-Arg-AMC as a preferred substrate for cathepsin B. Significantly, Z-Nle-Lys-Arg-AMC displayed the advantageous properties of measuring high cathepsin B specific activity over acidic to neutral pHs and was specifically cleaved by cathepsin B over the other cysteine cathepsins. Z-Nle-Lys-Arg-AMC specifically monitored cathepsin B activity in neuronal and glial cells which were consistent with relative abundances of cathepsin B protein. These findings validate Z-Nle-Lys-Arg-AMC as a novel substrate that specifically monitors cathepsin B activity over a broad pH range.

更新日期：2023-07-17

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