Profiling 26S Proteasome Activity of Plasmodium falciparum Monitored by a Live-Cell Assay

Malaria remains a major global health challenge, driven in part by widespread antimalarial drug resistance in <i>Plasmodium</i> parasites. Artemisinin-based combination therapies (ACTs) are currently the first-line treatment; however, resistance has also emerged. Artemisinin damages parasite proteins, promoting their ubiquitination and subsequent proteasomal degradation. Because inhibitors of the <i>Plasmodium</i> 26S proteasome synergize with artemisinin, the proteasome has emerged as a promising drug target, yet tools to monitor its function in live parasites remain limited. Here, we generated a <i>P. falciparum</i> line expressing green fluorescent protein fused to a destabilization domain (GFP-DD) to assess proteasome activity and combined it with MitoTracker^TM staining. In the absence of the stabilizing ligand Shield-1, the GFP-DD reporter is rapidly degraded by the proteasome. Using fluorescence microscopy and flow cytometry, we show that GFP-DD fluorescence provides a quantitative, inverse readout of proteasomal activity, increasing upon ligand-mediated stabilization or pharmacological inhibition with MG132. Shield-1 titration identified an optimal stabilization range, and MG132 induced a dose-dependent fluorescence increase. This work establishes a practical live-cell platform to probe ubiquitin-proteasome system function, with potential applications in future phenotypic screening and antimalarial resistance studies.