Oncode Institute & Department of Cell and Chemical Biology, Leiden University Medical Center, Einthovenweg 20, 2333 ZC Leiden, The Netherlands.
Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333 ZC Leiden, The Netherlands.
J Am Chem Soc. 2020 Sep 30;142(39):16825-16841. doi: 10.1021/jacs.0c07726. Epub 2020 Sep 18.
Many reagents have emerged to study the function of specific enzymes On the other hand, target specific reagents are scarce or need improvement, allowing investigations of the function of individual enzymes in their native cellular context. Here we report the development of a target-selective fluorescent small-molecule activity-based DUB probe that is active in live cells and an animal model. The probe labels active ubiquitin carboxy-terminal hydrolase L1 (UCHL1), also known as neuron-specific protein PGP9.5 (PGP9.5) and Parkinson disease 5 (PARK5), a DUB active in neurons that constitutes 1 to 2% of the total brain protein. UCHL1 variants have been linked with neurodegenerative disorders Parkinson's and Alzheimer's diseases. In addition, high levels of UCHL1 also correlate often with cancer and especially metastasis. The function of UCHL1 activity or its role in cancer and neurodegenerative disease is poorly understood and few UCHL1-specific activity tools exist. We show that the reagents reported here are specific to UCHL1 over all other DUBs detectable by competitive activity-based protein profiling and by mass spectrometry. Our cell-penetrable probe, which contains a cyanimide reactive moiety, binds to the active-site cysteine residue of UCHL1 in an activity-dependent manner. Its use is demonstrated by the fluorescent labeling of active UCHL1 both and in live cells. We furthermore show that this probe can selectively and spatiotemporally report UCHL1 activity during the development of zebrafish embryos. Our results indicate that our probe has potential applications as a diagnostic tool for diseases with perturbed UCHL1 activity.
许多试剂已经被开发出来用于研究特定酶的功能。另一方面，针对特定靶标的试剂却很缺乏或需要改进，从而无法在天然细胞环境中对单个酶的功能进行研究。在这里，我们报告了一种靶向选择性荧光小分子活性基 DUB 探针的开发，该探针在活细胞和动物模型中均具有活性。该探针标记活性泛素羧基末端水解酶 L1（UCHL1），也称为神经元特异性蛋白 PGP9.5（PGP9.5）和帕金森病 5（PARK5），UCHL1 是一种在神经元中活跃的 DUB，占大脑总蛋白的 1%至 2%。UCHL1 变体与神经退行性疾病帕金森病和阿尔茨海默病有关。此外，UCHL1 的高水平也常常与癌症特别是转移有关。UCHL1 活性的功能或其在癌症和神经退行性疾病中的作用尚未被充分了解，并且存在的 UCHL1 特异性活性工具也很少。我们表明，这里报道的试剂特异性地针对 UCHL1，而不是通过竞争性活性基蛋白谱分析和质谱可检测到的所有其他 DUB。我们的细胞穿透性探针含有氰基反应部分，以活性依赖的方式与 UCHL1 的活性位点半胱氨酸残基结合。其用途通过在活细胞中荧光标记活性 UCHL1 得到证明。此外，我们还表明，该探针可以在斑马鱼胚胎发育过程中选择性和时空报告 UCHL1 的活性。我们的结果表明，该探针具有作为 UCHL1 活性失调疾病的诊断工具的潜在应用。
