Structural and dynamical properties of central nervous system proteins with pharmaceutical and biotechnological potential.
Stanzione, Francesca (2010) Structural and dynamical properties of central nervous system proteins with pharmaceutical and biotechnological potential. [Tesi di dottorato] (Inedito)
Full text disponibile come:
Neurodegenerative diseases are widespread pathologies of large social impact that include: prion, Alzheimer and Parkinson disease, Huntington chorea and amyotrophic lateral sclerosis. The onset of such diseases is commonly associated with the accumulation of insoluble amyloid plaques in specific neuronal population. In this scenario, research activities for the prevention and the treatment of these diseases are focused on two distinct directions: (a) the enhancement of factors that promote the survival and maintenance of nerve cells and (b) the definition of the molecular processes that lead to onset of neurodegenerative diseases. In this framework, the main scopes of the present PhD project have been the analysis of structural/dynamic determinants of the function of neuroprotective proteins (neurotrophins) and the study of structural properties of amyloid aggregates and their toxic precursors. Neurotrophins (NTs) are homodimeric proteins that play a key role in the differentiation, survival and maintenance of nerve cells. This class of proteins include: nerve growth factor (NGF), Brain-Derived Factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), and neurotrophin 6 (NT6). NTs act by binding to two distinct classes of transmembrane receptors. One is the p75NTR neurotrophin receptor and the other is the Trk family of tyrosine kinase receptors, which includes TrkA, TrkB, and TrkC. All mature NTs bind to p75NTR, but Trks are more selective. NGF interacts selectively with TrkA receptors, NT4 and BDNF selectively with TrkB receptors, and NT3 interacts with TrkC receptors. During the PhD, a detailed investigation of the dynamical properties of different regions of NTs was carried out by molecular dynamics techniques. Initially, these studies were focussed on the intrinsic conformational preferences of N-terminal region of the NTs. These N-terminal regions are important for the recognition and the specificity of NT-Trk binding. Indeed, N-terminal region of NGF in complex with TrkA has an α-helical conformation, whereas the NT4 in complex with TrkB receptor is in 3/10 helix conformation. However, both N-terminal regions of the two NTs are absent in the crystallographic models of isolated dimers and in complex with the p75NTR receptor, revealing their flexibility in the absence of receptor and a conformational transitions in the interaction with the Trk receptor. Our calculations unveil that for NT4-Nter, and to a lesser extent for NGF-Nter, the conformation of the peptide that is prone to the Trk binding is already present among the states that are energetically accessible to the isolated peptide. This consideration has suggested feasible strategies for the design of effective NT agonist/antagonists. Indeed, variants of these peptides with an increased helical propensity will better mimic the NT functions. Successive simulations carried out on the main body of NGF have provided a detailed picture of mechanisms of interaction with the p75NTR receptor, whose stoichiometry of binding is controversial. These results provided important information on the correlated motions of distant region of the protein. Moreover, essential dynamics analyses clearly indicate that most of the motions of the protein are highly symmetrical. On the basis of results, it has been concluded that the binding of p75NTR to NGF induces a significant "induced-fit” from symmetric structures to asymmetric structures. In the last years, enormous efforts have been made to obtain insights into the structure of the amyloid-like forms of proteins and peptides involved in the insurgence of neurodegenerative diseases. A characterization of the structure and dynamic properties of these aggregates is required to define the molecular mechanisms underlying these diseases for the development of effective therapeutic strategies. In addition, the considerable resistance of amyloid-like fibrils, combined with their flexibility, versatility and ability to self-assemble has stimulated a growing interest in the potential of these fibers in biotechnological fields as nanobiomaterials. Previous MD simulations have shown that some steric zipper models are endowed with a remarkable stability also in a crystal-free context. However, MD simulations were limited to peptides with polar and/or aromatic dry interfaces. In this scenario, a section of my PhD project was focused on MD simulations of various amyloidogenic structures recently determined. Primarily, were carried out MD studies of steric zipper assemblies whose dry interface involves exclusively aliphatic residues. These simulations have highlighted the key role of residues involved in the steric zipper interface. Indeed, aliphatic residues are not able to form the intra-sheet and inter-sheet interactions formed by polar and aromatic residues that likely provide a strong contribution to the steric zipper motif stability. Along this line, amyoid-like assemblies endowed with hydrophobic residues presumably require larger interfaces, as it is shown by the stability of MD simulation of HET-s protein with a larger steric zippers interface. Very recent crystallographic studies have shown that the same amyloidogenic peptide can adopt distinct steric zipper assemblies (polymorphs). Intriguingly, it has been postulated that the different polymorphs of the same polypeptide sequence may be representative of distinct strains. In this framework, a detailed analysis of dynamical properties of two polymorphic structures formed by a segment of the islet amyloid polypeptide (IAPP) was carried out during the PhD. The analyses of the MD simulations show that the two IAPP distinct polymorphs are stable in a crystalline-free environment. This finding supports the hypothesis that the occurrence of strains in neurodegenerative diseases may be related to the possibility that a single peptide/protein chain may self-associate in alternative steric zipper-based assemblies. The last section of present thesis was dedicated to the studies of human prion protein (HPrP) properties. These studies were conducted in collaboration with the University of Cambridge. Independent crystallographic studies have shown the involvement of the β-sheet of the HPrP in intermolecular interactions that lead to the association of two different molecules HPrP in the crystalline state. These observations suggest that this association may be representative of the early stages of aggregation of HPrP. In this framework, during the PhD project detailed replica exchange molecular dynamics (REMD) studies on the intrinsic stability of HPrP β-structure were conducted. In particular, simulations were conducted on different β-strand combinations taken either from HPrP monomer or dimeric crystalline assemblies. The REMD simulations conducted on the isolated two stranded β-sheet of the protein monomer indicate that this structure is remarkably stable. The stability of larger aggregates formed by the juxtaposition of two of these sheets, as detected in the crystalline state, is very limited stability. Interestingly, additional simulations indicate that these aggregates are stabilized by mutations linked to the insurgence of pathological states. The observation that the two stranded β-sheet of the prion monomer are intrinsically stable hold important implications for prion polymerization process and for the design of synthetic peptides that potentially can inhibit the aggregation process of human prion protein.
Solo per gli Amministratori dell'archivio: edita il record