{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T09:09:28Z","timestamp":1772183368225,"version":"3.50.1"},"reference-count":24,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,2,24]],"date-time":"2026-02-24T00:00:00Z","timestamp":1771891200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001871","name":"Foundation for Science and Technology","doi-asserted-by":"publisher","award":["UIDB\/50026\/2020"],"award-info":[{"award-number":["UIDB\/50026\/2020"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Foundation for Science and Technology","doi-asserted-by":"publisher","award":["UID\/06304\/2025"],"award-info":[{"award-number":["UID\/06304\/2025"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Foundation for Science and Technology","doi-asserted-by":"publisher","award":["LA\/P\/0050\/2020"],"award-info":[{"award-number":["LA\/P\/0050\/2020"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Foundation for Science and Technology","doi-asserted-by":"publisher","award":["PTDC\/SAU-PAR\/2766\/2021"],"award-info":[{"award-number":["PTDC\/SAU-PAR\/2766\/2021"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Foundation for Science and Technology","doi-asserted-by":"publisher","award":["2020.02226.CEECIND"],"award-info":[{"award-number":["2020.02226.CEECIND"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001871","name":"Foundation for Science and Technology","doi-asserted-by":"publisher","award":["2022.12892.BD"],"award-info":[{"award-number":["2022.12892.BD"]}],"id":[{"id":"10.13039\/501100001871","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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\u2013proteasome system function, with potential applications in future phenotypic screening and antimalarial resistance studies.<\/jats:p>","DOI":"10.3390\/ijms27052104","type":"journal-article","created":{"date-parts":[[2026,2,24]],"date-time":"2026-02-24T13:53:20Z","timestamp":1771941200000},"page":"2104","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Profiling 26S Proteasome Activity of Plasmodium falciparum Monitored by a Live-Cell Assay"],"prefix":"10.3390","volume":"27","author":[{"given":"Adriana F.","family":"Gon\u00e7alves","sequence":"first","affiliation":[{"name":"Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal"},{"name":"Life and Health Sciences Research Institute (ICVS)\/Biomaterials, Biodegradables and Biomimetics Research Group (3B\u2019s)\u2014PT Government Associate Laboratory, 4710-057 Braga, Portugal"}]},{"given":"Ana","family":"Lima-Pinheiro","sequence":"additional","affiliation":[{"name":"Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal"},{"name":"Life and Health Sciences Research Institute (ICVS)\/Biomaterials, Biodegradables and Biomimetics Research Group (3B\u2019s)\u2014PT Government Associate Laboratory, 4710-057 Braga, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6580-0971","authenticated-orcid":false,"given":"Bel\u00e9m","family":"Sampaio-Marques","sequence":"additional","affiliation":[{"name":"Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal"},{"name":"Life and Health Sciences Research Institute (ICVS)\/Biomaterials, Biodegradables and Biomimetics Research Group (3B\u2019s)\u2014PT Government Associate Laboratory, 4710-057 Braga, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2682-7722","authenticated-orcid":false,"given":"Pedro E.","family":"Ferreira","sequence":"additional","affiliation":[{"name":"Life and Health Sciences Research Institute (ICVS), School of Medicine, University of Minho, 4710-057 Braga, Portugal"},{"name":"Life and Health Sciences Research Institute (ICVS)\/Biomaterials, Biodegradables and Biomimetics Research Group (3B\u2019s)\u2014PT Government Associate Laboratory, 4710-057 Braga, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2026,2,24]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (2024). 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