title: Label-Free and Homogenous Detection of Caspase-3-Like Proteases by Disrupting Homodimerization-Directed Bipartite Tetracysteine Display.
abstract: Caspase-3-like proteases (e.g., caspase-3 and caspase-7) constitute the core players of cell apoptosis, and their dysregulation has been linked to a number of human diseases, such as cancer and neurodegenerative disorders. However, the methods for measuring caspase-3-like proteases are often complex and time-consuming. Herein, we develop a label-free method to homogeneously detect caspase-3-like proteases in vitro and in complex cell lysate. This assay uses a modular peptide that contains a dimerization domain, a caspase-3/7 cleavage sequence, and a dicysteine motif as the activity sensor to detect caspase-3-like proteases. In the absence of caspase-3-like proteases, the homodimerization of modular peptide brings the dicysteine motif into close proximity and forms a particular configuration suitable for the binding to bisarsenical dye FlAsH-EDT2. The coordination of FlAsH-EDT2to dimeric peptide forms a highly fluorescent FlAsH-peptide complex. In contrast, the cleavage of the modular peptide by caspase-3-like proteases removes the dicysteine motif from the peptide and abrogates the bipartite tetracysteine display, leading to the disappearance of fluorescence. As a result, the caspase-3-like proteases can be quantitatively evaluated by measuring the change in fluorescence. This assay may be carried out in a "mix-and-read" manner and is, thus, quite simple and convenient. Moreover, this assay is extremely specific and can measure caspase-3 protein down to 1.28 × 10-4μg/mL. Importantly, the assay is compatible with complex biological samples and can measure both the activation and inhibition of intracellular caspase-3, thereby providing a new approach for the screening of caspase-targeted drugs and the diagnosis of apoptosis-associated diseases.
authors.affiliation: Laboratory for Food Safety and Environmental Technology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences , Shenzhen 518055, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Laboratory for Food Safety and Environmental Technology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences , Shenzhen 518055, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liang
authors.affiliation: College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University , Jinan 250014, China.
authors.forename: Chun-Yang
authors.identifier: 0000-0002-8010-1981
authors.initials: CY
authors.lastname: Zhang
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000032:analysis* / Q000378:metabolism; D053179:Caspase 7 / Q000032:analysis* / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine / Q000737:chemistry*; D019281:Dimerization; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry / Q000494:pharmacology; D051379:Mice; D000067996:RAW 264.7 Cells; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D053148:Caspase 3; D053179:Caspase 7; D003545:Cysteine