RICERCA ITALIANA     

Research program

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

University Co-ordinator

Università degli Studi di SIENA - ()

Research Unit Leader

Giuseppe Campiani

Description

Rationale. The link between oxidative stress and neuronal death is complex, but there is support for a contributory role of oxidative stress-induced neurotoxicity in neurodegeneration diseases such as AD. The condition of oxidative stress in this disease state is accompanied by a dyshomeostasis of metal ions, including the redoxactive transition metals iron and copper as well as redox inactive metal ions such as zinc. The central nervous system is particularly vulnerable to oxidative stress on account of the high rate of dioxygen utilization, the relatively poor concentrations of antioxidants and related enzymes, and the high content of polyunsaturated lipids, the most vulnerable biomacromolecule to oxidation. There is also an accumulation of iron in the brain as a function of age, which can be a potent catalyst for oxidative species formation. Metal ions that become separated from specific storage and transport proteins still readily coordinate adventitiously to proteins, nucleic acids, and circulating amino acids, so that the concentrations of “free” aqueous metal ions are very low. Accordingly redox inactive metal ions such as zinc may be pathogenic by virtue of their ability to displace redox-active metal ions from sites where redox activity of the latter is held in check. This does not mean, however, that only the “free” forms are active in ROS generation. As long as the coordination sphere of the metal ion is not saturated and supports the cycling between the oxidized and reduced states of the metal at biologically accessible potentials, the coordinated metal may serve as a catalytic center for generating ROS. The short diffusion distance of the main damaging ROS species, hydroxyl radical, suggests that most metal-catalyzed oxidative damage to proteins occurs via reaction of H2O2 (or perhaps O2) with the sites of metals coordinated adventitiously to the proteins. Examples of such “site-specific” oxidations, which have been modeled using an exogenous reducing agent and O2 or H2O2 as terminal oxidant include (i) conversion of His to 2-oxohistidine, (ii) oxidative coupling of Tyr, and (iii) oxidation of aromatic amino-acid side chains. Adventitious binding of transition metals to proteins can in many respects mimic redox metalloenzymes, though the rates are expected to be significantly slower. In this regard, although most scientists would consider adventitious protein-bound metals to exert pro-oxidant activity, anti-oxidant effects are thus also possible if these protein-metal systems mimic anti-oxidant metalloenzymes such as SOD or catalase. Proteins that serve to scavenge adventitious metal ions have been genetically engineered to abrogate the redox properties of the metal, such as is the case for the copper-binding site at the amino terminus of serum albumin. Proteins not normally functioning as metal-binding proteins may sometimes act as neuroprotectants by sequestering the metal ions in redox inactive forms. However, adventitiously-bound metals tend to have at least some redox catalytic activity, and it should be possible in some cases for adventitiously-bound metals to possess greater redox catalytic activity than the free metal ion. Furthermore, modification of proteins by carbonyl products of glycoxidation and lipoxidation can increase the capacity of the protein to bind copper and iron in a redox-active manner.
Six Tau isoforms are expressed depending on what isoforms of Tau are needed in particular locations of particular cell types or on what isoforms of Tau are needed in response to particular cellular events (such as in different stages of neuronal development or in reaction to stress). In the latter case, the cell may need to replace one Tau isoform with another in response to changing conditions; thus, it is possible that some phosphorylation of Tau is a normal cellular event regulated in order to specifically detach some of the Tau bound to microtubules in order to replace it with another isoform. If so, then the phosphorylated Tau would need to be degraded. If Tau became degradation-resistant or the degradation pathway failed, Tau would accumulate. Recent research has focused on evidence that dysfunctional proteasome activity, arising from inhibitory binding of PHF-tau, contributes to the intraneuronal accumulation of oxidatively damaged proteins in the brains of patients with AD, which may be sufficient to induce neuronal degeneration and death. In fact, defects of the ubiquitin-proteasome system lead to a condition known as “proteolytic stress” where the cell accumulates misfolded or abnormal (e.g., oxidatively modified) proteins that cannot be cleared. The inability of neurons to degrade abnormal protein is now a guiding principle affecting numerous neurodegenerative conditions, including AD, and can reflect interference with polyubiquitination as well as inhibition of the proteasomal pathway. In this view, it has to be recalled that the attachment of Ub to a target protein requires the action of three enzymes, E1 , E2 and E3, which work sequentially in a cascade. The E1 enzyme hydrolyses ATP and adenylates the C-terminus of UB, and then forms a thioester bond between the C-terminus of UB and the active site cysteine of E1. The E1 enzyme can then transfer the thioester-linked UB to the UB-conjugating enzyme, E2, in an ATP-dependent reaction. UB is linked by another thioester bond to the active site cysteine of the E2 enzyme. There are several different E2 enzymes (>30 in humans), are able to interact with overlapping sets of E3 ligases. With the help of E3, UB is transferred from the E2 enzyme to a lysine residue on a substrate protein. Additional UB molecules can be linked to the first one to form a poly-UB chain, which occurs through a particular type of E3 ligase sometimes referred to as a UB-elongation enzyme, or E4. There are seven lysine residues in UB that can be used to link UB molecules together, resulting in diverse structures. Poly-UB chains linked at different positions alters the destiny of the target protein to which it is added: Lys(11)-, Lys(29)- and Lys(48)-linked poly-UB chains target the protein to the proteasome for degradation, while Lys(6)- or Lys(63)-linked poly-UB chains (as well as mono-ubiquitinylation) signal reversible modifications in protein activity, location or trafficking. The length of the UB chain appears to be important as well, such as with Lys(48) poly-UB chains where its length influences its affinity for proteasomes.
Research Activity. The research unit UNISI has a great expertise in developing agents against neurodegenerative diseases and recently the research unit developed a new series of heterocyclic agents capable to selectively interfere with the amyloid/prion aggregate formation preventing amyloid fibrils from binding to each other (Compounds and Methods for Diagnosing and Treating Amyloid-related Conditions, PCT WO02/24652 A1), with metal chelating properties, and useful for in vivo imaging.Furthermore, UNISI scientists are skilled in protein/enzyme structural analysis, protein/protein interaction, homology building and medicinal chemistry.
On the basis of the previously obtained results, the aims of the present integrated project are:
1) Identification of putative low affinity transition metal binding sites on tau protein, ubiquitin, proteasome 26S, and E1, E2, E3 enzymes (bioinformatic analysis). Generation of computational models for a better understanding of the possible role of zinc, iron and copper in targeting the protein (quantum-chemstry approach). Design of experimental procedures aimed to optimize/validate the resulting computational model in collaboration with the other research units.
2) Analysis of the interaction of metal ions (iron, copper, zinc) with the identified low affinity interaction sites on the proteins, in relation to the different conformational stages of tau protein and UPS (molecular dynamics simulation studies). Integration of the experimental data, provided by UNICT and UNINA research units, into the molecular simulation procedure.
3) Generation of a 3D-pharmacophore model (3D-pharmacophore search approach) for the computer aided design of new specific molecules (Ub fragments, peptides and non-peptidic compounds) able to interfere with the metal-mediated inhibition of the Ub-proteasome pathway and ROS generation.
4) Synthesis of novel small molecules to be tested in cell free systems (UNICT, UNINA), in cells, and eventually in animal models by the research unit of Chieti, in order to identify innovative drugs to target conformational diseases.
To reach these goals it is essential to conduct an integrated research activity in collaboration with the other research units of this multidisciplinary research project. The data from computational analyses will be integrated with those obtained by NMR studies performed at UNINA, thermodynamic and spectroscopic investigations of complexes performed at UNICT and biological investigations performed by the research unit at Chieti University.
In particular, in the first phase of the project (objectives 1 and 2, months 1-8), research will proceed with the structural and bioinformatic analysis of a large number of experimentally determined structures of protein tau, Ub, and Ub related proteins, known or not to interact with metal ions, with the aim to identify adventitious transition metal ion interaction sites able to interfere with the UPS degradation of tau. For exemple, the beta amyloid protein possesses three histidine residues (His6, His13 and His14) located in the hydrophilic N-terminal part of the peptide and a methionine (Met35) residue in the lipophilic C-terminal region. These three residues have been identified as the crucial section involved with metal ion binding. Particularly, the histidine residues identify the site, which binds redox active Cu or Fe, while the methionine residue identifies the second site suggested to be involved in the reduction of Cu(II) and the generation of H2O2. A preliminary analysis of the primary structure of the proteins under investigation allowed us to hypothesize that adventitious binding of transition metal ions to low affinity protein binding sites may be involved also in the failure of UPS degradation of Tau. The interaction of metals with this protein sites would then cause oxidative damages which in turn would be responsible for UPS inihibition and for the precipitation of insoluble aggragetes of poy-ubiquitinated tau. At this purpose, it has to be underlined that the persistent insolubilization and permanency of neurofibrillary tangles (NFT) aggregates probably represents, at least in part, cementing of the aggregates by processes associated with oxidative stress. NFT display resistance to proteolysis, and this may reflect in part the presence of transaminase-mediated ?-glutamyl-e-lysine crosslinks among proteins identified in NFT (hsp27, SN, ubiquitin, parkin). Furthermore, NFT show a non-enzymatic redox activity.
We will particularly focus on the structural and computational analyses of Ub in complex or not with copper, zinc and iron ions, in order to allow the definition of the conformational changes of the protein in relation to the interaction with other proteins involved in UPS machinery. This theoretical analysis will drive the synthesis of specific protein fragments capable to interact with metal ions, mimicking the role of Ub (objective 3, months 6-12).
On the basis of the results obtained in the first phase of the project in collaboration with the research unit of Napoli (NMR investigation) and Catania (investigation of the stability of small molecules/metal complexes), the final goal of this research project will be the development a series of novel synthetic compounds with metal binding properties (zinc copper and iron), capable to cross the blood brain barrier, and to interfere with the structural and functional properties of transition metals low affinity biniding sites on small proteins, ipermetallated during AD. Metal ion selectivity will be also a major issue (objective 4, months 10-24).
The novel compounds to be developed within this project proposal should be characterized by low affinity (but high selectivity) for zinc, iron and copper ions, similar to specific brain proteins, in order to avoid metal depletion from brain and peripheral tissue (depletion is associated to the chelation therapy). The novel compounds should perfectly combine low metal affinity and selectivity with hydrophobicity. Hydrophobicity might allow the drug to penetrate the blood–brain barrier and once in the brain, the drug could potently affect the metal–protein equilibria, facilitating a correct functioning of the protein, inhibiting neurotoxic hydrogen peroxide production, and eventually the dissolution of aggregates. Hence, treatment withy these compounds would favour redistribution of the metals rather than excretion.
Thus, the novel compounds (heterocyclic or peptidic) will be designed to protect against zinc, iron and copper dependent neurotoxicity mainly mediated by ROS, and, in the case of peptidic compounds, with increased metabolic stability over carnosine. In vitro and in vivo studies (animal models of AD) (University of Chieti) may prove the beneficious effects of these compounds against cation and ROS neurotoxicity, being potentially beneficial in AD.
Materials and methods:
Bioinformatic analysis. The bioinformatic analysis will be performed using the softwares Biopolymer and Homology (Accelrys, San Diego) and the most known bioinformatic servers for proteins such as: i) ExPASy Proteomics Server, Swiss Institute of Bioinformatics, Gasteiger E et al., 2003, Nucleic Acids Res. 31:3784-3788, ii) InterDom 1.1, 2003, Institute for InfoComm Research, Singapore; iii) ProDom, 2005.1, INRA, CNRS, France, Catherine Bru et al., 2005, Nucleic Acids Res. 33: D212-D215; iv) Pfam 18.0, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK, Yeats and Sean R. Eddy 2004, Nucleic Acids Research, 32, D138-D141 24; v) ELM, elm.eu.org, Consortium funded by EU, Puntervoll P et al. 2003, Nucleic Acids Res., 31: 3625-3630.
Quantum Chemistry procedure. The evaluation of the chemical reactivity of the metal ions co-ordinated with the identified aminoacid mioety, the relative stability of the putative oxidation products, as well as of their transition states, will be evaluated performing semi-empirical (AMPAC/MOPAC, ZINDO) and ab-initio (Turbomole software package) calculations.
Molecular dynamics simulations. Molecular dynamics simulations will be performed using the modules Discover_3 and CharmM of the software package Insight2005 (Accelrys, San Diego). The dynamics run will be perfomed simulating different chemical environment (solvent, ionic strenght, pH values). Protein-protein docking will be performed using Affinity docking module. Results will be analysed using the module Analysis (Accelrys, San Diego).
3-D pharmacophore generation. A pharmacophore model of the key physical-chemical features required for compounds to compete with protein low affinity metal (iron, zinc and copper) binding sites wiil be developed (Catalyst, Accelrys, San Diego) and a 3D-Database search will be performed (Conquest 1.8, Isostar1.8 Cambridge Structural Database System Version 5.27) in order to find new chemical entities potentially endowed with different selectivity profiles. Particular attention will be paid to the calculation of the acidity dissociation constants of the newly designed compounds (ACD/pKa DB version 9.00, Advanced Chemistry Development Inc., Toronto, Canada). Compounds synthesis. Heterocyclic and peptide synthesis will be performed using standard organic synthesis methodologies or specifically developed synthetic pathways. Parallel synthesis will be applied to the synthesis of coherent sets of molecules.