The multifunctional human Parkinson’s disease protein 7 (PARK7/DJ1) is an attractive therapeutic target due to its link with early-onset Parkinson’s disease, upregulation in various cancers, and contribution to chemoresistance. However, only a few compounds have been identified to bind PARK7 due to the lack of a dedicated chemical toolbox. We report the creation of such a toolbox and showcase the application of each of its components. The selective PARK7 submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys106. Installment of different dyes onto the inhibitor delivered two PARK7 probes. The Rhodamine110 probe provides a high-throughput screening compatible FP assay, showcased by screening a compound library (8000 molecules). The SulfoCy5-equipped probe is a valuable tool to assess the effect of PARK7 inhibitors in a cell lysate. Our work creates new possibilities to explore PARK7 function in a physiologically relevant setting and develop new and improved PARK7 inhibitors.
SUMOylation is a reversible and highly dynamic post-translational modification of target proteins by small ubiquitin-like modifiers (SUMO). It is orchestrated by SUMO-activating, -conjugating, and -ligating enzymes in a sequential manner and is important in regulating a myriad of predominantly nuclear processes. DeSUMOylation is achieved by SUMO-specific proteases (SENPs). Deregulation of SUMOylation and deSUMOylation results in cellular dysfunction and is linked to various diseases, including cancer. In recent years, SENPs have emerged as potential therapeutic targets. In this review, we will describe the inhibitors and activity-based probes of SENPs. Furthermore, we will summarize the biochemical assays available for evaluating the activity of SENPs to identify inhibitors.
Expansion microscopy (ExM) has been widely used to detect biomolecules in cultured cells and tissue samples due to its enablement of super resolution imaging with conventional microscopes, via physical expansion of samples. However, reaction conditions inherent to the process bring about strong fluorescent signal loss during polymerization and digestion and thus limit the brightness of the signal obtained post expansion. Here, we explore the impact of stabilizer‐containing organic fluorophores in ExM, as a mitigation strategy for this radical‐induced dye degradation. Through direct conjugation of 4‐nitrophenylalanine (NPA) to our previously developed trifunctional reagents, we validate and demonstrate that these multifunctional linkers enable visualization of different organelles with improved fluorescent intensity, owning to protection of the dyes to radical induced degradation as well as to photoprotection upon imaging. At this point, we cannot disentangle the relative contribution of both mechanisms. Furthermore, we report anchoring linkers that allow straightforward application of NPA or Trolox to commercially available fluorophore‐conjugated antibodies. We show that these anchoring linkers enable complete retention of biological targets while increasing fluorophore photostability. Our results provide guidance in exploring these stabilizer‐modified agents in ExM and methods for increased signal survival through the polymerization steps of the ExM protocols.
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