UNIVERSITÀ DEGLI STUDI DI MILANO-BICOCCA
DOTTORATO DI RICERCA IN TECNOLOGIE CONVERGENTI PER I SISTEMI BIOMOLECOLARI (TeCSBi) – XXXIII CICLO
Project Supervisor: Dott. Paola Fusi
Project Title: Cadmium interference in human SOD1 structure and function: an in vitro model of neurodegenerative diseases
Possible support for Phd students w/o a University fellowship: none
Cadmium (Cd) is a widespread environmental contaminant, classified as a human non-genotoxic carcinogen (IARC 2012) which is primarily released into the environment by anthropogenic activities at a rate of ~30,000 tons/year. Release into the environment contributes to the metal introduction into the food chain and water. Once cadmium has entered the human body, generally through food, cigarette smoke and occupational exposure, it accumulates in various organs (primarily kidney, liver, lungs and reproductive organs) with a long biological half-life, estimated to be longer than 26 years. A Trojan horse strategy has been proposed for Cd toxicity, involving its uptake through zinc transporters and the interference with essential metals homeostasis, particularly with zinc. More specifically, cadmium-zinc exchange in proteins has been described as one of the main sources of Cd’s biological toxicity leading to increased free or labile zinc (Urani et al., 2015).
Human SOD1 is a 32-kDa homodimeric enzyme, in which each monomer binds one copper and one zinc atom; it catalyzes the dismutation of superoxide radical to oxygen and hydrogen peroxide. SOD1 mutations account for 20% familial amyotrophic lateral sclerosis (fALS) cases. Although SOD1 is one of the most thermally stable enzymes known in mesophilic organisms, zinc removal can destabilize the enzyme. Moreover, metal-depleted SOD1 has been implicated in the pathogenesis of fALS and mutations decreasing protein stability seem more likely to induce the disease (Lindberg et al. 2002).
Recent data obtained in our laboratory show that exposures of cultured neuronal cells to cadmium lead to a decrease in SOD1 activity and to an increase in oxidative stress. In this project, the effect of Zn substitution with Cd on human SOD1 structure and functionality will be studied through both a computational and a biochemical approach. We propose to produce a recombinant Cd-loaded human SOD1 (Cd-SOD1), whose activity, folding and propensity to aggregate will be assessed with respect to wild-type Zn-loaded SOD1. A bioinformatic study, conducted in parallel in collaboration with Prof. Maurizio Bruschi (DISAT Università di Milano Bicocca), will elucidate the mechanism of cadmium binding; results will be validated through site-directed mutagenesis. SOD1 mutants, produced on the basis of computational data, will be loaded with Cd in vitro and subjected to functional and structural characterization (including stability to temperature and denaturing agents, and propensity to aggregate).The same experiments will also be conducted on Cd-loaded SOD1 mutants most frequently associated with fALS,
Moreover, we propose to investigate Cd-SOD1 toxicity and outcomes in model cell lines, in collaboration with Dr. Chiara Urani (DISAT Università di Milano Bicocca). SOD1 functionality will be analyzed in cells grown in the presence of cadmium. Oxidative metabolism will be investigated mainly at the mitochondrial level, evaluating electron transport and ATP synthesis, membrane potential and coupling, both by biochemical methods and through confocal microscopy. Mitochondria morphology will also be investigated through confocal microscopy followed by quantitative imaging, while mitochondrial functionality will be assessed, through evaluation of Δψ , electron transport rate and its coupling to ATP synthesis. Reactive Oxygen Species (ROS) production will be assessed with specific fluorescent probes. The activity of enzymes involved in ROS detoxification, like glutathione peroxidase, glutathione reductase, glutathione S-transferase, catalase, glucose 6-phosphate dehydrogenase, will be assayed and their expression will be evaluated through real time PCR.
References
IARC, 2012. International Agency for Research on Cancer. A review on human carcinogens: arsenic, metals, fibers and dusts. IARC Monographs on the evaluation of carcinogenic risks to humans. 100C Lyon, France, pp. 121-141.
Lindberg MJ, Tibell L, Oliveberg M Common denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: decreased stability of the apo state. Proc Natl Acad Sci U S A 2002; 99: 16607–16612.
Urani C, Melchioretto P, Bruschi M, Fabbri M, Sacco MG, Gribaldo L, Impact of Cadmium on Intracellular Zinc Levels in HepG2 Cells: Quantitative Evaluations and Molecular Effects. BioMed Research International, 2015, 2015, 949514.