Structural plasticity in G-protein coupled receptors as demonstrated by the allosteric actions of homocysteine and computer-assisted analysis of disordered domains.

Structural plasticity of G-protein coupled receptors (GPCRs) is of basic importance for their interactions with ligands, in particular with other proteins such as receptors or receptor-modifying proteins that can lead to different functions for the same GPCR. In the present paper, structural plasticity of GPCRs has been investigated discussing allosteric modulatory actions of Homocysteine (Hcy) on D2 receptors together with data obtained by computer-assisted analysis of the presence of disordered domains in GPCRs. Previous evidence for a modulatory action of Hcy on D2 receptors has been further extended by means of experiments on the effects of Hcy local intrastriatal injection on rotational behaviour. Altogether the present data allow considering under a new angle the well known proposal of A2A antagonists as new therapeutic agents in Parkinson's disease (PD). Furthermore, they point out to not only the importance of drugs capable of reducing Hcy brain levels, but also to the potential therapeutic impact of drugs capable of regionally blocking (for PD) or enhancing (for some schizophrenic syndromes) Hcy allosteric action on D2 receptors. As far as the investigations on GPCR plastic domains, extracellular, intracellular and transmembrane domains of 14 GPCRs have been considered and propensity of each of these domains for a structured or unstructured conformation has been evaluated by means of ad hoc computer programs. It has been shown that the N- and C-terminals as well as intracellular loop 3 have a high propensity towards an unstructured conformation, hence they are potentially very plastic domains, which can undergo easily to interactions with other ligands, particularly with other protein domains. This aspect is obviously of the greatest importance not only for the function of single GPCRs, but also for their interactions either with other receptors (receptor-receptor interactions) or, more generally, for formation of clusters of membrane associated proteins, hence of "protein mosaics", where the GPCRs could represent the input unit of the supra-molecular device.

Role of cooperativity in protein folding and protein mosaic assemblage relevance for protein conformational diseases.

Biological systems are organized in intricate and highly structured networks with hierarchies and multiple scales. Cells can be considered as "meso-scale level" systems placed between the "macro-scale level" (systems of cellular networks) and the "micro-scale level" (systems of molecular networks). In fact, cells represent complex biochemical machineries made by networks of molecules connected by biochemical reactions. Thus, the brain should be studied as a system of "networks of networks". Recently, the existence of a Global Molecular Network (GMN) enmeshing the entire CNS was proposed. This proposal is based on the evidence that the extra-cellular matrix is a dynamic molecular structure capable of storing and releasing signals and of interacting with receptors and proteins on the cell membranes. Proteins have a special role in molecular networks since they can be assembled into high-order molecular complexes, which have been defined as Protein Mosaics (PM). Protein monomers in a PM (the "tesserae" of the mosaic) can interact via classical and non-classical cooperativity behaviour involving allosteric interactions. In the present paper, new features of allostery and cooperativity for protein folding, assemblage and topological features of PM will be discussed. Against this background, alterations in PM via allosteric modulations and non-classical cooperativity mechanisms may lead to protein aggregates like beta amyloid fibrils. Such aggregates cause pathological changes in the GMN structure and function leading to neurodegenerative diseases such as Alzheimer's disease. Thus, a novel view of the so called Protein Conformational Diseases (PCD) is proposed.

beta-Amyloid fibrillation and/or hyperhomocysteinemia modify striatal patterns of hyaluronic acid and dermatan sulfate: Possible role in the pathogenesis of Alzheimer's disease.

A key event in Alzheimer's disease (AD) pathogenesis is the formation of insoluble peptides beta-amyloid aggregates and this process is favoured by a condition of hyperhomocysteinemia. To date, there is growing evidence that implicates glycosaminoglycans (GAGs) in the pathophysiology of amyloidosis but no data are available on the characterization of brain GAGs involved in the enhancing beta-amyloid fibrillogenesis in relationship to their structure and physico-chemical properties. Furthermore, few studies have been performed on the relationship between hyperhomocysteinemia and extracellular matrix (ECM) modifications. The aim of this study was to evaluate the amount and chemical structure of GAGs in rat striatal areas where beta-amyioid fibrillogenesis was induced, and in conditions of hyperhomocysteinemia. The intrastriatal injection of beta-amyloid produced a significant decrease (-40.8%) in the hyaluronic acid (HA) percentage and an increase (+14.5%) in the dermatan sulfate (DS) with a total charge density increasing of 14.9%. A significant decrease (-19.5%) in the HA percentage and an increase (+6.9%) in the DS % was also observed in striata obtained from the hyperhomocysteinemic animals. The total charge density increased by 6.8%. Quite the same trend was observed in rats after intrastriatal injection of beta-amyloid and in a condition of hyperhomocysteinemia. The observed increase of DS concentration and the correspondent decrease of the nonsulfated polymer HA after in vivo treatment with beta-amyloid and in a condition of hyperhocysteinemia support the hypothesis that an increase in local production of sulfated GAGs may reduce beta-amyloid neurotoxicity. However, the consequent modification of the ECM network might impair the extracellular diffusion pathways of different signal molecules and participate in the progression of AD.

On the key role played by altered protein conformation in Parkinson's disease.

On the basis of the previously proposed hierarchic organisation of the central nervous system (CNS) and of its syntropic behaviour, a view of neurodegenerative diseases focusing on the assemblage of abnormal multimeric proteins (pathologic protein mosaics (PMs)) is proposed. Thus, the main focus of the present paper is on Parkinson's disease (PD) as a neurodegenerative disease, which has as crucial feature protein conformational alterations and formation of pathological PMs. Two interconnected cellular dysfunctions are discussed as main pathogenic factors of PD syndromes, namely mitochondrial deficits (i.e. energy failure, especially critical for Substantia Nigra DA neurons) and conformational protein alterations (due to genetic or environmental causes). Conformational protein alterations can trigger pathological phenomena via the loss and/or the gain of new functions. In particular, altered proteins can lead to the formation of abnormal PMs, which can, inter alia, cause distortion of cellular structures, toxic functions and/or formation of improper membrane ion channels. In view of the fact that disordered proteins can easily acquire unwanted conformation, the "disorder index" (DI) for proteins involved in PD has been evaluated. It has been found that both alpha-synuclein and tau-protein have high DI. This datum is in agreement with the observation that these two proteins synergistically promote polymerisation of each other into amyloid fibrils, favouring the formation of Lewy bodies.