Cross-platform multi-laboratory screening identifies HUWE1, USP33 and USP20 as negative-regulators of autophagy or mitophagy with relevance to neurodegeneration
- 1The ALBORADA Drug Discovery Institute, University of Cambridge
Abstract
Autophagy and mitophagy are essential cellular processes implicated in neurodegenerative diseases, yet few tractable targets have been robustly validated for therapeutic modulation of these pathways. In an industry-academia consortium we carried out an extensive literature review and expert curation, leading to selection of 29 genes previously reported to enhance autophagy upon genetic or pharmacological modulation. These genes were classified based on whether downregulation or overexpression induced autophagic activity. Using siRNA knockdown, small-molecule modulators, and transient transfection approaches, we systematically screened these targets in parallel across HeLa and HEK-293 cell lines using high- and low-content phenotypic imaging. Promising candidates were further evaluated in induced pluripotent stem cell (iPSC)-derived neurons and in Drosophila models. Three targets emerged as top candidates: HUWE1 downregulation consistently enhanced autophagy and aggregate clearance, while USP33 downregulation promoted mitophagy. We also identified USP20, a close homologue of USP33, as an inhibitor of mitophagy, a role not previously assigned to this protein. These effects on autophagy/mitophagy were corroborated in Drosophila, highlighting their potential functional relevance across species. This cross-platform, multi-laboratory study establishes a framework for identifying and validating regulators of autophagy and mitophagy and provides a route which enables a comprehensive reassessment of the current target literature in this field. The validation of HUWE1, USP20, and USP33 as candidate therapeutic targets offers new opportunities for intervention in neurodegenerative diseases.
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BibTeX
@misc{giudici2026crossplatform,
title = {{Cross-platform multi-laboratory screening identifies HUWE1, USP33 and USP20 as negative-regulators of autophagy or mitophagy with relevance to neurodegeneration}},
author = {M. L. Giudici and E. Coode and W. H. Au and C. Rey and M. Manifava and O.-G. Capatina and G. Aldred and S. Milde and V. Zyka and W.-L. Kuan and J. Duce and J. H. Clarke and A. J. Whitworth and N. T. Ktistakis and J. Skidmore},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.07.749806},
url = {https://www.biorxiv.org/content/10.64898/2026.09.07.749806v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Cross-platform multi-laboratory screening identifies HUWE1, USP33 and USP20 as negative-regulators of autophagy or mitophagy with relevance to neurodegeneration
AU - M. L. Giudici
AU - E. Coode
AU - W. H. Au
AU - C. Rey
AU - M. Manifava
AU - O.-G. Capatina
AU - G. Aldred
AU - S. Milde
AU - V. Zyka
AU - W.-L. Kuan
AU - J. Duce
AU - J. H. Clarke
AU - A. J. Whitworth
AU - N. T. Ktistakis
AU - J. Skidmore
PY - 2026
DA - 2026/09/07
PB - bioRxiv
DO - 10.64898/2026.09.07.749806
UR - https://www.biorxiv.org/content/10.64898/2026.09.07.749806v1
AB - Autophagy and mitophagy are essential cellular processes implicated in neurodegenerative diseases, yet few tractable targets have been robustly validated for therapeutic modulation of these pathways. In an industry-academia consortium we carried out an extensive literature review and expert curation, leading to selection of 29 genes previously reported to enhance autophagy upon genetic or pharmacological modulation. These genes were classified based on whether downregulation or overexpression induced autophagic activity. Using siRNA knockdown, small-molecule modulators, and transient transfection approaches, we systematically screened these targets in parallel across HeLa and HEK-293 cell lines using high- and low-content phenotypic imaging. Promising candidates were further evaluated in induced pluripotent stem cell (iPSC)-derived neurons and in Drosophila models. Three targets emerged as top candidates: HUWE1 downregulation consistently enhanced autophagy and aggregate clearance, while USP33 downregulation promoted mitophagy. We also identified USP20, a close homologue of USP33, as an inhibitor of mitophagy, a role not previously assigned to this protein. These effects on autophagy/mitophagy were corroborated in Drosophila, highlighting their potential functional relevance across species. This cross-platform, multi-laboratory study establishes a framework for identifying and validating regulators of autophagy and mitophagy and provides a route which enables a comprehensive reassessment of the current target literature in this field. The validation of HUWE1, USP20, and USP33 as candidate therapeutic targets offers new opportunities for intervention in neurodegenerative diseases.
ER -