Membrane-Targeted Quantum Dot-Based BACE1 Activity Sensors for In Vitro and In Cellulo Assays
Year: 2024
Type: article
Abstract: High Resolution Image Download MS PowerPoint Slide The need for the development of specific and robust methodologies to elucidate the intricate pathological mechanisms of neurodegenerative diseases and discover effective treatments for prevention and remediation is evident. Alzheimer’s disease, in particular, has become more prevalent as the global population has aged. β-Secretase, the β-site amyloid precursor protein cleaving enzyme (BACE1), is the protease that produces the β-amyloid peptide, which is considered one of the driving factors of Alzheimer’s disease and an important target for treatment development. However, an understanding of its activity, modulation, and regulation is far from complete. This is in large part due to the complex nature of following its activity. Beyond the common requirements for all biosensors (ease of preparation and use), BACE1 probes also demand both stability at acidic pH and membrane localization. To overcome these hurdles, we exploit the modular self-assembly provided by fluorescent quantum dot (QD) sensors. As compared to other fluorophores, QDs provide enhanced fluorescence brightness and photostability, and their large surface area enables functionalization with peptide substrates together with targeting elements that localize the sensor to the areas of maximal BACE1 activity, all achieved through His-tag self-assembly. In vitro, the sensor demonstrated stability under acidic conditions, and using high-throughput plate reader assays, we determined BACE1 activity in-line with literature values and enabled the obtainment of the inhibitor constant of verubecestat, a small molecule inhibitor. The sensor was also transitioned to cellular experiments, where it demonstrated sensitivity to BACE1 activity and its modulation upon inhibitor treatment in a neuroblastoma cell line.
Institutions: Consejo Superior de Investigaciones Científicas, Instituto de Salud Carlos III, Biomedical Research Networking Center on Neurodegenerative Diseases, Centro de Investigación Biomédica en Red, Centro de Investigaciones Biológicas Margarita Salas, IMDEA Nanoscience, United States Naval Research Laboratory, Naval Research Laboratory Center for Bio/Molecular Science and Engineering, Centro Nacional de Biotecnología
Language: English
Subfield: Physiology
Field: Medicine
Domain: Health Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
Funders: European Commission, Ministerio de Ciencia e Innovación, Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Ministerio de Educación y Formación Profesional, Instituto de Salud Carlos III, Agencia Estatal de Investigación, Office of Naval Research, U.S. Naval Research Laboratory, European Social Fund, Office of Naval Research Global
OpenAlex ID: W4404169934