Publications by authors named "Marta Massari"

3 Publications

  • Page 1 of 1

A lonely electron blocks incoming pairs.

J Biol Chem 2021 Jan-Jun;296:100294. Epub 2021 Feb 12.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Pavia, Italy. Electronic address:

Electron bifurcation exploits high energetic states to drive unfavorable single electron reactions and determining the overall mechanism governing these electron transfers represents an arduous task. Using extensive stopped-flow spectroscopy and kinetic simulations, Sucharitakul et al. now explore the bifurcation mechanism of the electron transfer flavoprotein EtfAB from the anaerobic gut bacterium Acidaminococcus fermentans. Strikingly, they illustrated that catalysis is orchestrated by a negatively charged radical, α-FAD, that inhibits further reductions and features an atypical inverted kinetic isotope effect. These results provide additional insight behind electron transfers that are prevalent within multienzyme governed reactions.
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http://dx.doi.org/10.1016/j.jbc.2021.100294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948957PMC
July 2021

A closer look into NADPH oxidase inhibitors: Validation and insight into their mechanism of action.

Redox Biol 2020 05 15;32:101466. Epub 2020 Feb 15.

Department of Biology and Biotechnology "Lazzaro Spallanzani", University of Pavia, Via Ferrata 9, 27100, Pavia, Italy. Electronic address:

NADPH-oxidases (NOXs) purposefully produce reactive-oxygen-species (ROS) and are found in most kingdoms of life. The seven human NOXs are each characterized by a specific expression profile and a fine regulation to spatio-temporally tune ROS concentration in cells and tissues. One of the best known roles for NOXs is in host protection against pathogens but ROS themselves are important second messengers involved in tissue regeneration and the modulation of pathways that induce and sustain cell proliferation. As such, NOXs are attractive pharmacological targets in immunomodulation, fibrosis and cancer. We have studied an extensive number of available NOX inhibitors, with the specific aim to identify bona fide ligands versus ROS-scavenging molecules. Accordingly, we have established a comprehensive platform of biochemical and biophysical assays. Most of the investigated small molecules revealed ROS-scavenging and/or assay-interfering properties to various degrees. A few compounds, however, were also demonstrated to directly engage one or more NOX enzymes. Diphenylene iodonium was found to react with the NOXs' flavin and heme prosthetic groups to form stable adducts. We also discovered that two compounds, VAS2870 and VAS3947, inhibit NOXs through the covalent alkylation of a cysteine residue. Importantly, the amino acid involved in covalent binding was found to reside in the dehydrogenase domain, where the nicotinamide ring of NADPH is bound. This work can serve as a springboard to guide further development of bona fide ligands with either agonistic or antagonistic properties toward NOXs.
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http://dx.doi.org/10.1016/j.redox.2020.101466DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042484PMC
May 2020

Engineering stability in NADPH oxidases: A common strategy for enzyme production.

Mol Membr Biol 2017 May - Dec;34(3-8):67-76. Epub 2019 Jan 10.

a Department of Biology and Biotechnology , University of Pavia , Pavia , Italy.

NADPH oxidases (NOXs) are membrane enzymes whose sole function is the generation of reactive oxygen species. Humans have seven NOX isoenzymes that feature distinct functions in immune response and cell signaling but share the same catalytic core comprising a FAD-binding dehydrogenase domain and a heme-binding transmembrane domain. We previously described a mutation that stabilizes the dehydrogenase domain of a prokaryotic homolog of human NOX5. The thermostable mutant exhibited a large 19 °C increase in the apparent melting temperature (app T) and a much tighter binding of the FAD cofactor, which allowed the crystallization and structure determination of the domain holo-form. Here, we analyze the transferability of this mutation onto prokaryotic and eukaryotic full-length NOX enzymes. We found that the mutation exerts a significative stabilizing effect on the full-length NOX5 from both Cylindrospermum stagnale (app T increase of 8 °C) and Homo sapiens (app ΔT of 2 °C). Enhanced thermal stability resulted in more homogeneous preparations of the bacterial NOX5 with less aggregation problems. Moreover, we also found that the mutation increases the overall expression of recombinant human NOX4 and NOX5 in mammalian cells. Such a 2-5-fold increase is mainly due to the lowered cell toxicity, which leads to higher biomasses. Because of the high sequence identity of the catalytic core within this family of enzymes, this strategy can be a general tool to boost the production of all NOXs.
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http://dx.doi.org/10.1080/09687688.2018.1535141DOI Listing
January 2019
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