Mossuto, Maria Francesca (2008) Protein amyloidogenesis: characterization of aggregation prone conformations and fibrils structure. [Ph.D. thesis]
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Current interest in studying amyloid fibrils arises from their involvement in different fields (Chiti and Dobson, 2006). First, they play a crucial role in disorders such as Alzheimer's and Parkinson's diseases. Second, since it has been demonstrated that all polypeptide chains form fibrils under appropriate conditions, the understanding of why and how this process happens has become central problem in protein knowledge. Last, the ordered ultrastructure characterizing amyloid fibrils may be thought as a basis for nanomaterials with possible technological applications. However, despite the ability of most proteins to form amyloid fibrils, very little is known about their structures and the factors that govern their formation.
The process of amyloid formation requires the partial unfolding of protein molecules into such conformations able to interact to each others and reorganize into well-ordered structured aggregates, named amyloid fibrils.
In this Thesis the amyloid formation by globular proteins has been analyzed from different points of view, focusing in the first part of the research work on the elucidation of some conformational features promoting protein aggregation. The second part was concentrated on the characterization of the final supramolecular structure of amyloid fibrils.
In order to study the partially folded state, two globular proteins have been analyzed, alpha-lactalbumin (LA) and HypF-N. Indeed, under specific conditions, these proteins populate a not fully folded state previously shown to play an important role in the amyloid formation (Uversky, 2002; Chiti et al., 2001).
The study conducted on LA has been based on the effects of the proteolytic dissection of the molecule on its conformational features and aggregation properties. It was previously shown that LA is able to form fibrils morphologically indistinct from the pathological ones (Uversky et al., 2002). Here, we have studied the aggregation propensities of LA derivatives characterized by a single peptide bond fission (1-40/41-123, named Th1-LA) or a deletion of a chain segment of 12 amino acid residues located at the level of the ?-subdomain of the native protein (1-40/53-123, named des?-LA). We have also compared the early stages of the aggregation process of these LA derivatives with those of intact LA. The main conclusion of this work was that the inherent flexibility of the LA derivatives allows the large conformational changes required to form the cross-?-structure of the amyloid fibrils. It has been emphasized that proteolysis can be considered a causative mechanism of protein aggregation and fibrillogenesis (Polverino de Laureto et al., 2005).
In the other case, the conformational characterization of an amyloidogenic state of HypF-N has been performed at acid pH, in order to allow the protein to populate a partially unfolded ensemble. Combining different biophysical and biochemical techniques, it has been shown that this partially unfolded structure has all the hallmarks of a pre-molten globule state, i.e. it is more compact than a random coil-like state but less organized than a native-like intermediate or a MG state (Uversky, 2002). Furthermore, it is shown that a modulation of the total ionic strength of the solution allows enhancing the apparent rate of aggregation of HypF-N under these conditions. This increased rate of aggregation has been shown to be mediated by the interaction of monomers to form initial oligomers, through a particular region in the sequence, corresponding to the sequence part having highly hydrophobicity, the highest beta-sheet propensity and with no net charge at acid pH, representing the ideal segment suitable to mediate protein oligomerization.
From all these studies, it is clear that, except the unique native state of globular proteins wherein the side chains pack together in a unique manner, every state of a polypeptide molecule is a broad ensemble of often diverse conformations. It is not surprising, therefore, that even the fibrillar products of aggregation processes are characterized by morphological and structural diversity, representing variations on a common theme.
The second part of my PhD Thesis deals with the structural characterization of fibrils. Many studies have been conducted on amyloid aggregates formed under different conditions by peptides, such as A?, TTR and prion fragments (Kodali and Wetzel, 2007). Indeed, the problem of amyloid formation by a full-length protein is more complex, since the dense packing reachable in amyloid fibrils made of peptides (10-40 residues) could not be accomplished in all the amino acid residues of a full-length protein, except in the core regions (Chatani and Goto, 2005). The object of my study was human lysozyme due, most of all, to the fact that some natural variants of human lysozyme (HuL) are responsible for the formation of amyloid plaques in vivo, in a so called familial non-neuropathic systemic amyloidosis (Pepys et al., 1993; Booth et al., 1997). Moreover, it has been possible to exploit the available wealth of structural and folding information about wild type HuL, since it has been shown to be able to form fibrils quite similar to the pathological ones, under acidic conditions and high temperature (Morozova-Roche et al, 2000). With respect to fibrils made of peptides, besides, studying amyloid fibrils conformations from HuL is more challenging because it is a 130 amino acid chain with the structural constrains given by the four disulfide bridges present in the lysozyme molecule. This study can also give some insights into the complex problem of strains diversity, such as for prion diseases, helping the clarification of the structural principles of amyloid fibrils which can produce multiple and distinct amyloid conformations from one protein sequence.
In the presented study, fibrils of wild-type HuL formed at low pH have been analyzed by limited proteolysis experiments and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates (Frare et al., 2006). After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive ?-sheet structure and substantial elements of non ?-sheet or random structure that are reduced significantly in the fibrils after digestion. The sequence 32-108 includes the ?-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme (Frare et al., 2004). The present data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain.
HuL variants, however, aggregate in a physiological environment, roughly at pH 7-7.5 at 37 °C, because of their instability (Dumoulin et al., 2005). In my work, it has been demonstrated that also HuL is able to aggregate under conditions similar to the pathological ones, presumably neutral pH and 37 °C. Considering that HuL forms amyloid fibrils in such different conditions (pH 2.0 50°C and pH 7.5, 60°C), a comparison of the structure and the stability of fibrils obtained under these different conditions has been conducted. In this study HuL fibrils were produced at acidic and at neutral pH, leading both to the formation of fibrils having the three hallmarks of amyloid, that are cross-beta structure, binding of ThT and an overall amyloid fiber morphology. These fibrils have been studied by means of ANS binding, FTIR and X-ray fiber diffraction in order to characterize the differences in the structure. Guanidinium-induced fibrils dissociation, instead, has been applied in order to test the chemical stability of the two kinds of fibrils. The results clearly indicate that the solution conditions used for lysozyme aggregation promote the formation of fibrils with different structural features and stability properties, due to the diverse rearrangements of the lysozyme polypeptide chain into the fibril structure.
In conclusion, the research work conducted in this Thesis allowed the comprehension of important aspects of the unfolding of some globular proteins leading to amyloid fibrils. In addition, original data have been obtained on the structural polymorphism of amyloid fibrils.
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