RICERCA ITALIANA     

Research program

Role of metals – Ubiquitin/Proteasome interaction in the pathogenesis of conformational diseases

University Co-ordinator

Università degli Studi di CATANIA - SCIENZE CHIMICHE - ()

Research Unit Leader

Enrico Rizzarelli

Description

According to preliminary studies carried out in our lab (D. Grasso, D. Milardi, A. Magrì, G. Maccarrone, C. La Rosa, G. Tabbì, E. Rizzarelli, Interaction of metals with Ubiquitin: a possible interference in proteins turnover? 1 European conference on Chemistry of Life Science, Rimini, Italy October 4-8 2005) some metals may decrease the thermal stability of the Ubiquitin. This evidence prompted us to plan further investigation specifically aimed at establishing the stoichiometry, the binding sites, the coordination geometries and the stability of the metal-protein complexes. At first, we will assess if specific metals are able to bind to the Ubiquitin, and consequently, to modify its thermodynamic stability and structure. In particular, Differential Scanning Calorimetric (DSC) studies will be carried out on Ubiquitin/metal solutions in order to study the effect of pH, ionic strength and protein:metal molar ratio on the thermodynamic stability of Ubiquitin. Moreover, we will investigate the metal binding sites by means of ESI-MS, Circular Dichroism (CD), 2D Nuclear Magnetic Resonance (NMR), Electron Spin Resonance (ESR) and UV-Vis spectroscopy. These studies are expected to provide information about the structures of the different metal complexes with protein which will be eventually refined by Molecular Dynamics (MD) simulations. Because of the expected low solubility of Ubiquitin/metal complexes, specific and reliable model peptides will be synthesized in order to quantitatively assess their affinities for the different metals by means of potentiometric methods. The stability constants will be compared with other metal binding proteins, in an attempt to find possible pathogenic molecular mechanisms involving metal-trafficking. CD and NMR studies will be also performed in order to describe the metal-induced modifications of Ubiquitin conformation. Furthermore, the effect of redox-active metal ions as Cu and Fe, on some specific target residues as Met1 will be investigated by ESI-MS. It is known, that Reactive Oxygen Species (ROS) may oxidize Met 1 thus giving rise to several protein isomers with different thermodynamic stability and structure. Met 1 is hydrogen – bonded with Lys 63 and it is thus foreseeable that an oxidation of Met 1 may have consequences in the impairment of polyubiquitination mechanisms involving Lys 63. Oxydized Ubiquitin isomers will be analyzed by ESI-MS, DSC, CD and NMR. The determination of the structural features of metal/Ubiquitin complexes will be carried out in collaboration with the Research Unit coordinated by Prof. Di Blasio. In collaboration with the research Unit coordinated by Prof. G. Campiani, the assessment of the binding features of different metal ions on Ubiquitin will be exploited to design a library of molecules which may antagonize harmful metal binding,
Molecular studies on metal / Ubiquitin complexes will be paralleled by experiments aimed at assessing the effects of metal ions, particularly copper and zinc, on primary cultures of rat cortical neurons. It is known that zinc and copper ions can be endogenous modulators of neuronal excitability and that they have the ability to affect the main hydrolitic activities of the purified 20S proteasome. Cultured cortical neurons will be challenged with different concentrations of zinc (as ZnCl2) or copper (as either Cu(II)-glycine or CuSO4) for different lengths of times. Each metal will be added to sufficiently low concentrations to obtain neurotoxic effects with long times of exposure (36-72 hrs). Metal-induced toxicity will be assessed by means of MTT assay, and p53 induction will be verified as a marker for neuronal apoptosis either by immunocitochemistry or western blot analysis. Whole cell extracts from control and metal-treated cultures, will be utilized to measure proteasome activity. Proteasome activity assay will be carried out by using fluorogenic substrates for the chymotrypsin-like, the trypsin-like and the caspase-like actvity of the 20S particle. Fluorogenic substrates for all known proteasome activity are available from BIOMOL International, and they will be used to determine whether metals affect a specific proteasome activity or rather produce a global proteasome impairment. In addition, the effects of zinc and copper will be compared to those exerted by lactacystin, a proteasome inhibitor endowed with neurotoxicity. Provided that proteasome activity is reduced by either one or both metals, the accumulation of ubiquitylated proteins will be verified by western blot analysis of total protein extracts and by confocal microscopy imaging of ubiquitin intra-neuronal inclusions (ubiquitin-specific antibodies are commercially available). Time courses of proteasome activity and ubiquitin immunoreactivity will be carried out within the time frame to complete metal-induced neurotoxicity and to determine whether the build-up of ubiquitylated proteins follow proteasome inhibitions. If metal ions interfere initially with protein ubiquitylation process, a reduced amount of ubiquitin coniugates in the western blot analysis of metal-treated neurons will be observed. Regulation of 20S proteasome activity occurs at several levels. Proteasome activity is triggered by the association of the 20S complex with two 19S regulatory subunits. The 19S complex provides the different regulatory functions that are necessary to ensure selective degradation of ubiquitin-tagged substrates. Therefore, we will investigate whether metal ions are able to impair proteasome activity by interfering with the mechanisms of 26S proteasome assembly/disassembly. In particular, co-immunoprecipitation studies will be carried out on total neuronal extracts to demonstrate the lack of associaton between the catalytic β subunit of the 20S particle with Rpn10, the ubiquitin receptor subunit of the 19S particle. Alternatively, subcellular colocalization studies of the different proteasome subunits (antibodies are commercially available) will be performed by confocal microscopy. Studies on cell models will be carried out in collaboration with the research unit coordinated by Prof. Sensi.
After the characterization of the different metal ions effects on the several partners involved in the ubiquitine-proteosoma system UPS, we will try specific metal chelators as potential therapeutical agents of the UPS. At first we will study the effect of carnosine, a dipeptide with known chelating and antioxidant properties, on the different steps of the metal-loaded UPS. Moreover, several carnosine derivatives that cannot be cleaved by carnosinases present in the brain, will be tested both in cell-free systems and in rat cortical neurons. Finally, molecules designed on the basis of the chemical studies on Ubiquitin-metal complexes will be also tested.
In collaboration with Dr. Luigi Zecca, the Catania Research Unit will address the effect of the components of neuromelanin-iron complex on the activity of 26S proteasome. These components may inhibit the activity of 26S proteasome and determine the selective vulnerability of dopamine neurons in ageing and related disorders such as Parkinson Disease (PD). In order to study this potential effect, brain samples will be collected during autopsies of male and female subjects without evidence of neuro-psychiatric and degenerative disorders. Samples of substantia nigra will be carefully dissected and tissues for histology and histochemistry evaluation will be fixed in formalin and processed accordingly to search for Lewy bodies and other pathological markers. Then the neuromelanin will be isolated from human substantia nigra. Because the activity inhibition of 26S proteosoma can be related to the low affinity iron bound to neuromelanin or to other components of neuromelanin structure, three different components of neuromelanin-Fe will be isolated by using suitable experimental protocol:1) lipid free neuromelanin; 2) neuromelanin with cleaved peptide chain; 3) neuromelanin at variable iron content These specific components will be manipulated to prepare derivatives for in vitro and in situ studies to establish which aspects of the neuromelanin molecule are responsible for proteasome inhibition.
Furthermore different types of model melanins will be synthesized by autoxidation using precursors including dopamine and cysteine, and employed for the same purpose.