Εξειδίκευση τύπου :	Άρθρο σε επιστημονικό περιοδικό
Τίτλος:	Optimization of a High-Throughput Screen for Monitoring Disease-Associated Protein Misfolding and Aggregation in Bacteria
Δημιουργός/Συγγραφέας:	Delivoria, Dafni C Konia, Eleni Matis, Ilias [EL] Σκρέτας, Γιώργος[EN] Skretas, George
Ημερομηνία:	2025-05-12
Γλώσσα:	Αγγλικά
ISSN:	2161-5063 2161-5063
DOI:	10.1021/acssynbio.5c00166
Άλλο:	40354780
Περίληψη:	Protein misfolding and aggregation are central features of a wide range of diseases, including neurodegenerative disorders, systemic amyloidoses, and cancer. The identification of compounds that can modulate protein folding and aggregation is a key step toward developing effective therapies. High-throughput screening methods are essential for efficiently identifying such compounds. In this study, we optimized a previously developed high-throughput genetic screen for monitoring protein misfolding and aggregation in bacteria. This system is based on monitoring the fluorescence of Escherichia coli cells expressing fusions of human misfolding-prone and disease-related proteins (MisPs) with the green fluorescent protein. We systematically tested a variety of experimental conditions, such as overexpression conditions and MisP-GFP fusion formats, to identify key parameters that affect the sensitivity and dynamic range of the assay. Using misfolding-prone, cancer-associated variants of human p53 as a model system, we found that strong overexpression conditions, such as high copy number vectors, strong promoters, high inducer concentrations, and high overexpression temperatures, can yield optimal assay performance. These optimized assay conditions were also validated with additional MisPs, such as the Alzheimer's disease-associated amyloid-β peptide and variants of superoxide dismutase 1 associated with amyotrophic lateral sclerosis. At the same time, we observed that certain conditions, such as inducer concentrations and overexpression temperature, may need to be precisely fine-tuned for each new MisP target to yield optimal assay performance. Our findings provide a framework for standardizing MisP-GFP screening assays, facilitating their broad application in the discovery of therapeutic agents targeting protein misfolding and aggregation.
Τίτλος πηγής δημοσίευσης:	ACS synthetic biology
Θεματική Κατηγορία:	[EL] Βιοχημεία[EN] Biochemistry [EL] Μοριακή Βιολογία[EN] Molecular Biology [EL] Γενετική[EN] Genetics [EL] Βιοτεχνολογία[EN] Biotechnology
Λέξεις-Κλειδιά:	SOD1 amyloid β green fluorescent protein high-throughput screening p53 protein aggregation protein misfolding diseases
Χρηματοδότης:	European Research Council (ERC) HellenicFoundation for Research and Innovation (HFRI General Secretariat for Research and Technology (GSRT)
EU Grant:	European Union’sHorizon 2020 ProMiDis Twin4Promis Boost4Bio HFRI PhD Fellowship
EU Grant identifier:	81993 101079363 10108747 19345
Κάτοχος πνευματικών δικαιωμάτων:	© 2025 The Authors. Published by American Chemical Society
Όροι και προϋποθέσεις δικαιωμάτων:	This article is licensed under CC-BY 4.0.,
Ηλεκτρονική διεύθυνση στον εκδότη (link):	https://doi.org/10.1021/acssynbio.5c00166
Εμφανίζεται στις συλλογές:	Ινστιτούτο Χημικής Βιολογίας - Επιστημονικό έργο