A molecular platform for intracellular nitric oxide sensing

Nitric oxide (NO) exerts multiple biological functions through autocrine and paracrine signaling pathways, particularly in the regulation of vascular tone, neurotransmission, acute and chronic inflammation, and host defense against pathogens, including viruses, bacteria, and fungi. Following real-time NO fluctuations in concentration at the single-cell level is a challenging task due to its low concentration, short lifetime, and high reactivity with a variety of reactive oxygen species.
Only a few genetically encoded fluorescent sensors (GES) have been so far developed and proposed for monitoring intracellular NO levels, but their complexity, large size and multi-domain structure raise concerns about their reliability. These limits highlight the need for the development of sensitive yet simpler GES for NO monitoring.
This project exploits our seminal observation that the blue-emitting fluorescent protein mTagBFP2, undergoes a reduction in fluorescence quantum yield and lifetime upon exposure to μM NO concentrations, an effect that has been correlated with S-nitrosylation of Cys residues.
Taking advantage of a multidisciplinary team with internationally-recognized expertise in chemical physics of biological systems, protein design, and engineering, time-resolved spectroscopy, and fluorescence microscopy, this project proposes to explore fluorescent proteins (FP) as simple NO sensors to obtain a molecular platform of GES suitable for fluorescence imaging, including fluorescence lifetime imaging (FLIM). 

PNRR-M4C2- I1.1- Avviso MUR n. 1409 del 14-09-2022 – Bando PRIN 2022 PNRR - Settore ERC PE4 Titolo Progetto: A molecular platform for intracellular nitric oxide sensing - Codice Progetto P2022F4WR8 - Codice CUP D53D23016840001 - Finanziato dall’Unione Europea – NextGenerationEU

Università di Parma (UNIPR): Cristiano Viappiani (national coordinator), Stefania Abbruzzetti, Stefano Bruno, Pietro Delcanale, Manuela Maria Alampi, Lucia Bellanova

Istituto Nanoscienze - Consiglio Nazionale delle Ricerche, Pisa (CNR-NANO): Riccardo Nifosì (unit coordinator)

Università di Catania (UNICT): Salvo Sortino (unit coordinator), Venera Cardile

• August 27-29, 2024: Results of the project presented at the 10th Regional Biophysics Conference (RBC2024). Part of the recent results of the project were communicated at the international meeting “10th Regional Biophysics Conference (RBC2024)” by Cristiano Viappiani in an oral communication entitled Molecular basis for the nitric oxide sensitivity of a blue fluorescent protein (PDF abstract). This lecture reported the findings of the collaborative research between the group at the University of Parma and the CNR-NANO group in Pisa. The experimental and theoretical activity involved also scientists working at the Forschungszentrum Jülich, Germany. Besides the numerous senior scientists, the work has seen the involvement of two master students and one PhD student.
  The combination of experimental studies and molecular modelling allowed to identify a plausible mechanism at the basis of the nitric oxide sensitivity of fluorescence emission by mTagBFP2 and other fluorescent proteins.

   

• June 16-20, 2024: our PhD student Lucia Bellanova wins the prize for the best poster at the XXVII National Congress of the Italian Society of Pure and Applied Biophysics (SIBPA), in Genoa, for her work Sensing nitric oxide with a fluorescent protein.

   

• April 4, 2024: The group of Prof. Sortino develops an ultrafast, turn-on nitric oxide fluorescent sensor. A nitric oxide (NO) probe based on a BODIPY fluorogenic center covalently linked to a trimethoxy aniline appendage has been developed. NO leads to effective nitrosation of the amine binding site, resulting in an increase of the fluorescence of the BODIPY by almost one order of magnitude accompanied by significanty changes in the amplitude of the fluorescence lifetimes. This probe exhibits a response time < 0.1 s, high sensitivity, responsiveness also to peroxynitrite, independence of the fluorescence response in a wide range of pH, good selectivity towards different analytes and small interference by typical physiological concentration of glutathione. 

  Parisi, C.; Pastore, A.; Stornaiuolo, M.; Sortino, S. A fluorescent probe with an ultra-rapid response to nitric oxide. J. Mat. Chem. B 2024, DOI: 10.1039/D4TB00064A.

• First results presented at the international scientific meeting Focus on Microscopy - FOM2024, Genoa, Italy, Sunday March 24 to Wednesday March 27, 2024; Poster presentation P1-G / 13 - Sensing nitric oxide with a fluorescent protein L. Bellanova, K. Morreale, A. Franzen, B. Santiago-Schübel, S. Bruno, A. Scarano, S. Abbruzzetti, P. Delcanale, C. Viappiani, T. Gensch (abstract link)