| Autor: |
Tosat-Bitrián C; Centro de Investigaciones Biológicas 'Margarita Salas'-CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain.; Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas, (CIBERNED), Instituto de Salud Carlos III, Av. Monforte de Lemos, 3-5, 28029 Madrid, Spain., Bueso de Barrio JA; Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia), C/Faraday 9, 28049 Madrid, Spain., Stewart MH; Optical Sciences Division, Code 5600, U.S. Naval Research Laboratory, Washington, DC 20375, United States., Susumu K; Optical Sciences Division, Code 5600, U.S. Naval Research Laboratory, Washington, DC 20375, United States., Medintz IL; Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, United States., Díaz SA; Center for Bio/Molecular Science and Engineering, Code 6900, U.S. Naval Research Laboratory, Washington, DC 20375, United States., Palomo V; Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas, (CIBERNED), Instituto de Salud Carlos III, Av. Monforte de Lemos, 3-5, 28029 Madrid, Spain.; Instituto Madrileño de Estudios Avanzados en Nanociencia (IMDEA Nanociencia), C/Faraday 9, 28049 Madrid, Spain.; Unidad Asociada al Centro Nacional de Biotecnología (CSIC), Darwin 3, 28049 Madrid, Spain. |
| Abstrakt: |
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. |
