RICERCA ITALIANA     

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

Università degli Studi di Catania

Abstract

It’s now recognised that several socially relevant neurodegenerative diseases, such as Alzheimer’s, Parkinson and Prion diseases, share the same pathogenic mechanism. Indeed, in each of these pathologies an abnormal increment of insoluble protein deposits, often referred to as “amyloids” can be found. Despite intense scientific effort has been addressed, during the last decades, to the comprehension of the molecular events underlying these pathologies, several unclear aspects have still to be unravelled before an effective therapy could be proposed. Yet, it has been demonstrated the determining role of several ageing-related factors, particularly the oxidative stress generated by ROS or the metals-promoted amyloidogenesis in consequence of perturbation, at a cellular level, of metal ions homeostasis.
Increasing experimental evidence suggests that in several pathologies linked to aberrant protein conformation, the normal protein turn-over inside the cell is inhibited. In Eukaryotes, physiological protein elimination inside the cell, is controlled by the Ubiquitin-Proteasome System (UPS), a complicated network of enzymatic reactions that, by using the ubiquitin as an identification tag for those protein destined to elimination and proteasome as the proteolytic machinery, degrades them into lower molecular weight fragments. Recent papers have demonstrated that divalent metal ions, (Cu2+, Fe2+ and Zn2+) including some red-ox active ones, can act as UPS inhibitors, thus holding for the attracting hypothesis of a common molecular pathway connecting metal-unbalancing, oxidative stress, protein aggregation, UPS inhibition and neurodegeneration.
Although some aspects of such a complex picture are known, some others and not less important, must be explored. In particular, no any systematic study has been yet undertaken to investigate on metal ion interaction with the different UPS components.
Through a multidisciplinary approach, the present research project intends to contribute to fill up this gap by exploiting and merging the specific competences of the four research units. By conjugating the chemical characterization of the metal complexes generated with the different UPS components and the biological analysis in cellular and animal models, the comprehension and rationalization of several unknown mechanisms can be expected. This in turn may provide a possible platform for the future design of more appropriate therapeutic strategies.
In particular by means of NMR, CD, ESI-MS, ESR, DSC and potentiometry, the chemical-physics and thermodynamic parameters, characterizing ubiquitin/metal ion interactions and related partners, will be determined. This will allow the construction of an useful model to be utilized for the creation, by means of virtual screening, of a library of hypothetical antagonist molecules capable of restoring the normal UPS activity previously endangered by metal ions action. Studies on cellular cultures will be carried out to identify and characterize specific steps of the proteolytic process that are liable to different metals inhibition. The analyses performed on cellular cultures will be completed with in vivo experiments carried out on transgenic rats, as well as on post-mortem human tissues samples. These will be executed by using imaging techniques. Such a “scale-up process” from the molecule to the living organism will provide at the same time an exhaustive amount of helpful data for the complete knowledge of the phenomena underlying the pathogenic process and an useful protocol for testing the therapeutic potential of the new antagonist molecules that will be synthesized on the basis of the acquired results. <<<

Principal Investigator

Enrico Rizzarelli Università degli Studi di CATANIA

Research Objectives

The ubiquitin-proteasome system (UPS) has emerged as a predominant cellular regulatory machinery with roles in controlling cell-division, signal transduction, development and the immune response. It is also implicated in many neurodegenerative diseases, since cellular aggregates characterising neurodegeneration are heavily ubiquitinated. Despite the intense efforts aimed to elucidate the role played by UPS system in all these cellular processes, the mechanisms of protein regulation systems within the brain are poorly understood. Consequently, a long-term research goal is to understand protein regulation in the brain, and their implications in neurodegeneration and cell-cycle control. It is well known that the age-dependent rise in the brain of metal ions might contribute to hypermetallate many proteins, thus triggering misfolding and precipitation. However, the role played by metals in the disfunction of the ubiquitin-proteasome system is not completely understood. The present research project is aimed to investigate about the possibility that metal ions have to interact with the UPS and, as a consequence, to interfere with its normal activity. In this ligth, efforts will be mainly focused to elucidate the mechanisms by which components of the UPS may affect protein regulation and cell-cycle control within the brain. Dissection of the molecular mechanisms controlling protein turnover will not only enhance our understanding of these critical systems, but will also provide scaffolds for the design of novel therapeutics to target conformational diseases.

Specific targets of the project are:
i) To elucidate the binding site, the coordination features, and the affinity of metal ions to Ubiquitin, thus providing a reliable molecular model for a better understanding of the possible role played by metal ions in targeting different regions of the protein.
ii) To assess the effects of metal binding on the stability and structure of Ubiquitin, and their possible consequences on the downstream processes involving polyUbiquitination and specific activation of different cellular processes.
iii) To put in evidence, the disturb caused by different metal ions on the proteolytic activities of the proteasome.
iv) To distinguish between metal-driven oxidative processes and proteolysis inhibition pathways able to cause overload of different proteins
v) To provide, on the basis of molecular studies, a solid framework for computer-aided design of new specific molecules able to interfere with the metal-mediated inhibition of UPS.
iv) To test the activity of newly synthesized molecules in cell-free systems, in cells and finally in animal models in order to screen drugs with a promising potential for eventual pre-clinical and clinical trials. <<<

Timescale

24 months

National and international background

Several disorders, named "Conformational Diseases" [1], including the prevalent dementias and encephalopathies, are now believed to share the same general pathogenic mechanism. In each, there is an abnormal accumulation of insoluble aggregates that usually consist of fibrils containing a misfolded protein in a beta-sheet conformation, termed amyloid. The gradual accumulation of these aggregates and the acceleration of their formation by stressful environmental factors explain the characteristic late or episodic onset of the clinical symptoms. The understanding of these processes at the molecular level is opening prospects of more rational approaches to investigation and therapy [1]. There is partial but not perfect overlap among the cells in which misfolded proteins are deposited and the cells that degenerate. The most likely explanation is that inclusions and other visible protein aggregates represent an end stage of a molecular cascade of several steps, and that earlier steps in the cascade may be more directly tied to pathogenesis than the inclusions themselves [2]. There is no evident sequence or structural homology among the proteins that have been implicated in protein conformational disorders. However, there is accumulating evidence that the aggregates formed by the different misfolded proteins have the same molecular form.
Because of their insolubility and non-crystalline nature, high-resolution studies of aggregated proteins have been difficult. But recent studies using X-ray fibre diffraction and solid-state nuclear magnetic resonance have confirmed the beta-sheet-rich structure of protein aggregates implicated in neurodegenerative diseases. These studies have resulted in a molecular model of amyloid-like fibrils composed of several protofilaments, which consist of hydrogen-bonding beta-sheet structures with the beta-strands running perpendicular to the long fibre axis, a structure known as a cross-beta conformation. It is clear from these structural studies that a large conformational rearrangement of the polypeptide chain occurs during misfolding and aggregation. However, it is not known whether the misfolding triggers protein aggregation or protein oligomerization induces the conformational changes. On the basis of the available evidence, it is likely that slight conformational changes result in the formation of a misfolded intermediate, which is unstable in an aqueous environment because of the exposure of hydrophobic segments to the solvent. This unstable intermediate is stabilized by intermolecular interactions with other molecules, forming small oligomers which, with further growth, produce amyloid-like fibrils. According to this model, the conversion of the folded protein into the pathological form is triggered by structural changes, but complete misfolding depends on oligomerization. Environmental factors that might catalyse protein misfolding include changes in concentration of metal ions, pathological chaperone proteins, pH or oxidative stress, macromolecular crowding and increases in the concentration of the misfolding protein. Many of these alterations are associated with ageing, consistent with the late onset of neurodegenerative diseases [3]. An increasing number of observations indicate that transition metals, in their di- and trivalent ionic form, are capable of accelerating the aggregation process of various pathologic proteins, e. g. a-synuclein ( a-syn), the amyloidbpeptide (A b), b2-microglobulin ( b2-m) and fragments
of the prion protein (PrP) [4-9]. In particular, there are two generic reactions of relevance to these diseases. Firstly, a metal-protein associations leading to protein aggregation; this reaction may involve redox-inert metal ions such as Al, Zn, or redox-active metal ions such as Cu, Fe, and Mn [6, 9]. Secondly, metal-catalyzed protein oxidation leading to protein damage and denaturation; this reaction involves a redox active metal ion [8, 10,11].
Growing evidence indicates that failure to eliminate misfolded proteins can lead to the formation of potentially toxic aggregates, inactivation of functional proteins, and ultimately cell death. The number of disease states linked to aberrant protein conformations underscores the importance of effective quality control for cell survival [12]. In eukaryotic cells, the ubiquitin–proteasome systems (UPS) is the main pathway for eliminating misfolded proteins [13-14] and for this fundamental discovery Ciechanover, Hershko and Rose have been awarded for Nobel Prize in 2004. As a result, blocking its function pharmacologically or genetically inhibits the clearance of misfolded proteins and eventually leads to the formation of intracellular aggregates. Proteins are earmarked for UPS-mediated degradation by the covalent attachment of a polyubiquitin chain(s), which is recognized by the 26S proteasome. Only substrates targeted to the proteasome by polyubiquitination are able to gain access to its proteolytic core. Ubiquitination is a multi-step process involving an E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin ligases, which select the substrate and facilitate ubiquitination. Polyubiquitination of some proteins also requires so-called E4 enzymes that cooperate with E3 ligases to extend the polyubiquitin chain [15]. Many aggregation-based diseases reflect a failure of the UPS quality control system, either in surveillance or in elimination, and an imbalance between protein synthesis, folding and degradation. The link between the ubiquitin–proteasome system and neurodegeneration has been strengthened by the observation that inclusion bodies and other proteinaceous aggregates within affected cells often contain Ubiquitin [16]. Besides functioning in protein quality control, the ubiquitin–proteasome pathway also regulates a number of cellular processes through the degradation of specific proteins and Ubiquitin itself might also be involved in regulation of protein function, in addition to turnover. [17]. Indeed, the ubiquitin–proteasome pathway has been implicated in a growing number of human diseases, and represents a promising therapeutic target in certain cancers [18,19]. An important question in understanding UPS activity concerns how this wide functional range is achieved. Polyubiquitin (polyUb) chains — polymers formed through Ub–Ub conjugation — occur within cells, can be linked to target proteins, and take on diverse structures due to the presence of seven lysine residues in Ub. The existence of structurally distinct polyUb chains could represent one way to enhance diversity in Ub-dependent signalling [20]. Because the functional outcome of polyubiquitination can be linkage (structure) specific, determination of the residues and structural features involved in chain formation is important. Function-relevant features of Ub’s compact 3D structure include a hydrophobic surface patch formed by L8, V70 and I44, and a flexible C-terminus ending with G76. A combination of the hydrophobic effect and the electrostatic potential caused by the positive charges (on K6, R42, K48, H68 and R72, surrounding the hydrophobic patch) are likely to be important for Ub’s interaction with multiple factors. This leads to the possibility of self-interaction among units in the chain, and the hydrophobic patch L8–V70–I44 indeed mediates intra-chain interactions between Ub units that determine the conformation of certain polyUb chains. Whether close hydrophobic contacts can occur between Ub units depends on the location of the linkage lysine relative to Ub’s hydrophobic patch. Linkage-dependent differences in polyUb conformations could be functionally important because they result in a differential display of the surface hydrophobic residues implicated in Ub-dependent signaling events. As an example, K63-linked chains are now known to signal in four pathways: DNA damage tolerance [21], the inflammatory response [22], protein trafficking [23], and ribosomal protein synthesis [24]. Besides activating enzymes, environmental factors such as pH variations, ionic strength, metals and other chaperone molecules are also believed to play a not negligible role in controlling the UPS pathway [20]. These evidences, coupled with the demonstration that the failure of the ubiquitin-proteasome system is involved in many neurodegenerative disorders prompted many people to examine which factors may mainly contribute to a general impairment of the proteolytic machinery. Moreover, it is well known that the age-dependent rise in the brain of metal ions, in particular Cu and Zn. This event might contribute to hypermetallate many proteins, thus triggering oxydative stress, misfolding and precipitation [8]. Oxidatively –induced accumulation of ubiquitinated proteins in mouse neuronal cells has been yet demonstrated and suggests that the UPS pathway is closely involved in the cell response to metal-induced oxidative stress [25]. Furthermore, recent studies concerning the effect of oxidative stress induced by neurotoxic metal ions on the properties of the 20S proteasome have demonstrated that exposure of the 20S proteasome to increasing amounts of Fe(III), Fe(II), Cu(II) or Zn(II) affects its main hydrolytic activities: trypsin-like (T-L), chymotrypsin-like (ChT-L), peptidylglutamyl-peptide hydrolase (PGPH), branched-chain amino acid preferring (BrAAP) and caseinolytic activities, although in different ways. The functional effects appear to be linked to oxidation-induced modifications, as demonstrated by an increase of carbonyl groups following the exposure to metal ions [26].
Taken all together, these results prompted a number of studies aimed to elucidate the potential therapeutical use of different metal-interacting molecules targeting the UPS pathway. In particular, it has been found that treatment of pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, resulted in the accumulation of several proteasome substrates. The PDTC effect was due to an extended half-life of these proteins through the mobilization of zinc. PDTC and/or zinc also increased fluorescence intensity of UbG76V-GFP fusion protein that is degraded rapidly by the ubiquitin-proteasome system. Treatment of cells with zinc induced formation of ubiquitinated inclusions in the centrosome, a histological marker of proteasome inhibition. Western blotting showed zinc-induced increase in laddering bands of polyubiquitin-conjugated proteins. In vitro, Zinc inhibited the ubiquitin-independent proteasomal degradations of a-synuclein [27]. It has been reported that organic copper complexes can potently and selectively inhibit the chymotrypsin-like activity of the proteasome in vitro and in vivo. In particular, bis-8-hydroxyquinoline copper(II) [Cu(8-OHQ)2], was able to inhibit the chymotrypsin-like activity of purified 20S proteasome. Furthermore, it has been found that copper-mediated inhibition of purified 20S proteasome cannot be blocked by a reducing agent and that organic copper compounds do not generate hydrogen peroxide in the cells. This suggests that proteasome inhibition and apoptosis induction are not due to copper-mediated oxidative damage of proteins. These results suggest that certain types of organic ligands could form potent proteasome inhibitors in presence of copper whose action mechanism is different from oxidation [28]. In addition, it has been recently reported that clioquinol (CQ), an analog of 8-hydroxyquinoline and an Alzheimer's disease drug, and pyrrolidine dithiocarbamate (PDTC), a known copper binding compound and antioxidant, can interact with copper to form cancer-specific proteasome inhibitors and apoptosis inducers in human breast cancer cells [29]. It has also been demonstrated that expression of a dominant-negative mutant form of ubiquitin (K48R) in NT-2 and SK-N-MC cells caused decreased cell growth rates and increased oxidative damage (protein carbonyls and lipid peroxidation), nitric oxide production and elevated protein nitration [30]. In dopaminergic neurons of substantia nigra and norepinephrine neurons of locus coeruleus neuromelanin plays an important role in the homeostasis of metals and especially binds large amount of iron [31- 32]. These neurons containing neuromelanin undergo selective neurodegeneration in Parkinson’s disease while other neurons are spared [33]. An increase of total iron content was found in substantia nigra during aging and Parkinson’s disease [31-34]. A specific accumulation of the complex neuromelanin-iron occurs in dopaminergic neurons of substantia nigra during aging [35]. Neuromelanin can reduce the activity of 26S proteasome, as shown in situ using a green fluorescent protein homologue targeted to 26S proteasome and also in vitro using ubiquitinated lysozyme as a substrate. Neuromelanin reduces the amount of PA700 regulatory subunit of 26S proteasome, but does not affect that of a- and b-subunits of 20S proteasome. However it is not clear which component of neuromelanin structure like quinone, lipid, peptide or metals is truly cytotoxic and inhibit proteasome activity [36].
Conclusively, although there are many tantalizing clues that the ubiquitin–proteasome pathway is crucial for neurodegenerative disease pathogenesis [37], determination of the specific mechanisms and confirmation in vivo remain at now elusive goals. In part, this is because we still lack a clear understanding at a molecular level of how misfolded proteins are targeted for ubiquitination and degradation. Otherwise, we need to define clearly what factors may interfere with the cascade of molecular events involved in the Ubiquitin-Proteasome pathway. Understanding the details of these mechanisms will be important for developing novel therapeutic interventions. The metal-mediated proteasome inhibition hypothesis is elegant and could provide a unifying mechanism for diseases involving protein misfolding. <<<