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

## TL;DR

This study introduces a live-cell assay to measure proteasome activity in malaria parasites, which could help in developing new treatments.

## Contribution

A novel live-cell assay using GFP-DD and MitoTracker to monitor proteasome activity in Plasmodium falciparum.

## Key findings

- GFP-DD fluorescence increases with proteasome inhibition or stabilization, providing an inverse readout of activity.
- MG132 induces a dose-dependent increase in fluorescence, confirming its inhibitory effect on the proteasome.
- The assay offers a practical platform for studying the ubiquitin–proteasome system in live parasites.

## Abstract

Malaria remains a major global health challenge, driven in part by widespread antimalarial drug resistance in Plasmodium 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 Plasmodium 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 P. falciparum line expressing green fluorescent protein fused to a destabilization domain (GFP-DD) to assess proteasome activity and combined it with MitoTrackerTM 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.