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Novel fluorescently labelled ATP analogues for direct monitoring of ubiquitin activation
摘要： Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real-time are scarce. To address this shortcoming, we developed novel fluorescently labelled adenosine tri-, tetra- and pentaphosphate analogues of ATP. The novel ATP analogues bear – in contrast to earlier reports – only a single acridone-based dye at the terminal phosphate group. The dye’s fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP cleaving enzymes can be followed in real-time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin-activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate-harbouring ATP analogue supersedes the efficiency of recently reported dual-dye labelled analogues and thus, is a promising candidate for broad applications.
关键词： ubiquitin,fluorescent probes,PET,UBA1,ATP
更新于2025-09-19 17:13:59
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Azo-PROTAC: novel light-controlled small-molecule tool for protein knockdown
摘要： Reversibly altering endogenous protein levels are persistent issues. Herein, we designed photoswitchable Azo-PROTACs by including azobenzene moieties between ligands for the E3 ligase and the protein of interest. Azo-PROTACs are light-controlled small-molecule tools for protein knockdown in cells. The light-induced configuration change can switch the active state to induce protein degradation activity, which can be reversely controlled by light exposure in intact cells. We compared the protein degradation abilities of Azo-PROTACs with different configurations and linker lengths. Using the stable form with the best degradation ability against the BCR-ABL fusion and ABL proteins in myelogenous leukemia K562 cells, we showed that Azo-PROTAC combines the potent protein knockdown and facile cell uptake properties of the small-molecule PROTAC with a reversible photo-switch ability, offering a promising chemical knockdown strategy based on the light-induced reversible on/off property.
关键词： protein modifications,photoswitchable PROTAC,protein knockdown,ubiquitin-proteasome system.
更新于2025-09-19 17:13:59