Dissertation committee:
Βασιλική Οικονομίδου, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Ισιδώρα Παπασιδέρη, Ομότιμη Καθηγήτρια, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Ευαγγελία Χρυσίνα, Κύρια Ερευνήτρια, Ινστιτούτο Χημικής Βιολογίας, Εθνικό Ίδρυμα Ερευνών
Ιωάννης Τρουγκάκος, Καθηγητής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Ευστάθιος Καστρίτης, Καθηγητής, Τμήμα Ιατρικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Εμμανουήλ Μικρός, Καθηγητής, Τμήμα Φαρμακευτικής, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Μαριάννα Αντωνέλου, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
Soluble proteins must fold into their normal three-dimensional (3D) structure in order to function properly. However, under certain conditions, proteins may fail to adopt or maintain their normal conformation, and thus may undergo general or partial refolding of their polypeptide chain. Misfolded proteins may interact with each other, leading to the formation of either amorphous or highly ordered protein aggregates. The latter share distinctive structural and tinctorial properties and are called amyloid fibrils, while proteins able to form them are known as “amyloidogenic proteins”. Uncontrolled extracellular and/or intracellular deposition of amyloid fibrils in cells and tissues is the cause of a variety of diseases, known as amyloidoses. Typical examples of these pathologies are AL and AA amyloidosis, Alzheimer's and Parkinson's disease, spongiform encephalopathies and type II diabetes. Taking into account their major role in disease, it is striking that many organisms – ranging from bacteria to humans – exploit the properties of amyloid fibrils to perform physiological functions necessary for their survival. These structures are characterized as protective or functional amyloids and one of their most characteristic representatives is the protective coats enclosing the oocytes of many eukaryotic organisms.
Amyloid-forming proteins do not share any similarity or homology in sequence or native structure. However, amyloids – whether formed in vivo or in vitro – exhibit common morphological features and a similar "general" organization, known as the "cross-β" structure. In addition, not all regions of a polypeptide chain appear to have the same importance for amyloid fibril formation. More specifically, experimental evidence has shown that certain amino acid regions display higher aggregation propensity than the rest of the sequence. These short “aggregation-prone” stretches are called “aggregation hot-spots” or “amyloidogenic determinants” and are considered responsible for the self-aggregation of proteins associated with amyloidoses and with the formation of functional amyloids.
This thesis focused on the experimental study of “aggregation-prone” regions of a heterogeneous set of proteins, with known or unknown amyloidogenic properties. The amyloidogenic segments were computationally identified and experimentally evaluated with the aid of several biophysical techniques, such as transmission electron microscopy, X-ray diffraction, polarizing microscopy, fluorescence assay and ATR-FTIR spectroscopy, at a range of conditions. In addition, computational studies were performed utilizing molecular modeling, to propose plausible polymerization models for each protein. Furthermore, structural bioinformatics and protein-protein interaction networks were utilized to explain the possible amyloidogenic functional role of each protein.
Calcitonin gene-related peptide (CGRP) exists in the human body in two isoforms, αCGRP and βCGRP. αCGRP is the predominant form and shows high degree of homology with amylin and calcitonin, two proteins that have been extensively associated with the formation of amyloid fibrils, causing type II diabetes and medullary thyroid carcinoma, respectively. In view of the well-established calcitonin and IAPP amyloidogenicity, the aggregation properties of the full-length αCGRP as well as that of three computationally determined “aggregation-prone” regions were examined, utilizing both in vitro aggregation assays and Molecular Dynamics (MD) simulations. Our experimental assays detected characteristic amyloidogenic properties for the full-length αCGRP peptide hormone, whereas analogous experiments tracked the hidden amyloidogenic characteristics of N- and C- terminal regions of αCGRP. MD simulations, on the other hand, led to the proposal of two alternative polymerization pathways, with the experimentally confirmed amyloidogenic determinants serving as critical molecular interfaces by mediating the self-association between adjacent αCGRP protofibrils.
Human leukocyte chemotactic factor-2 (LECT2) is a multifunctional protein, produced by the fetal and adult liver and secreted in the blood. Apart from its numerous functional properties, LECT2 has been associated with various pathological conditions, such as cancer and diabetes. This protein has been relatively recently associated with a systemic type of amyloidosis, namely ALECT2 amyloidosis. Although, the pathogenesis of this disease remains to be elucidated, it does not appear to be caused by the identified – in almost all ALECT2 patients – polymorphism at position 40 of the mature amino acid sequence, which is responsible for the conversion of the amino acid residue isoleucine (Ile) to valine (Val). In this thesis, we followed a reductionist methodology to detect critical amyloidogenic “hot-spots” during the fibrillation of LECT2. By associating experimental and computational assays, the impact of the Ile to Val single-nucleotide polymorphism in patients with ALECT2 amyloidosis was discussed, pinpointing the need for additional research. The results of this work revealed the explicit amyloidogenic core of human LECT2. A possible three-dimensional model, associating our experimental and computational results, establishes LECT2 interactions that may occur as amyloidogenesis proceeds.
Cathepsin D (CathD) is a lysosomal aspartic protease, important for the degradation of various substrates, including in vivo amyloid-forming proteins. More specifically, CathD plays a key role in the degradation of serum amyloid A protein, while it is the main protease in the brain and can degrade the microtubule-associated protein tau. To clarify the potential implication of CathD in amyloid formation, aggregation-prone regions were detected in the full-length protein. The computational analysis of its amyloidogenic profile allowed the design of oligopeptides corresponding to the amyloidogenic CathD core, as well as “cropped” peptides, corresponding to regions that surpass the threshold of the used algorithm. Biophysical assays revealed that all peptides self-assemble into aggregates with the typical characteristics of amyloid fibrils, which demonstrate a wide morphological polymorphism at a range of conditions. In addition, our results hint that the respective aggregation-prone CathD regions may be implicated in its interaction with its amyloidogenic substrates.
The plant natriuretic peptide of Arabidopsis thaliana (AtPNP-A) contains a region of 34 amino acid residues, known as AtPNP-A36-69, which is a key component of its biological function. This region is conserved among all members of the PNPs family and has a distant homology with the human ANP (hANP). The latter forms amyloid fibrils and their deposition is the major hallmark of isolated atrial amyloidosis (IAA). Taking into consideration the well-established amyloidogenicity of hANP, along with the functional similarity to its plant counterpart, we decided to investigate the unexplored polymerization properties of the homologous region that corresponds to the biologically active and conserved functional domain of AtPNP-A. Our results revealed a new case of a pH-dependent amyloid forming peptide in A. thaliana, with a potential functional role.
FimH, a mannose-specific adhesin, is found on the tip of Escherichia coli type 1 fimbria or pili, the adhesive pili on the surface of the bacterial cell. FimH is responsible for the occurrence of urinary tract infections, since its interaction with urothelial receptors facilitates the establishment and colony formation of E. coli and subsequently causes human immune evasion, resulting in recurrent infections. The most common treatment for this type of diseases is the short-term administration of antibiotics. However, antibiotic-resistant strains have increased in recent years, intensifying the need for new therapeutic approaches. Thus, this thesis attempted to utilize the amyloidogenic potential of the FimH adhesin to suggest potential antimicrobial peptides. Computational analysis of its sequence revealed several possible amyloidogenic determinants, from which four peptide segments were selected for the study. These four peptides are located near the mannose-binding pocket of the N-terminal lectin domain of FimH. The experimental results demonstrated the ability of three of the four peptide-analogues to self-assemble into amyloid fibrils. Furthermore, a computational comparison proving the FimH peptide-analogues uniqueness among proteomes emphasized the potential of using those peptides as antimicrobial peptide-inhibitors, aiming to eliminate urinary tract infections. Finally, with the aid of structural bioinformatics, the affinity of the four peptide-analogues for the FimH mannose-binding site was examined, proposing an additional therapeutic approach.
The silkmoth chorion is a proteinaceous structure that plays a significant role both in protecting the embryo from environmental hazards and the fertilization of the silkmoth oocyte. The major part (95% of its dry mass) of this extraordinary structure consists of proteins categorized into two families, namely, A and B. Silkmoth chorion has been identified as a natural protective amyloid, by unveiling the amyloidogenic properties of the central domain of both protein families. In addition, during their secretion, it is assumed that the proteins of the A and B families interact and, subsequently, form fibrils that are further organized to form the chorion structure. Based on the above, the aim of this study was to clarify the mechanism of silkmoth chorion formation by studying the interactions between amyloidogenic peptide-analogues of the central conserved domain of the silkmoth chorion proteins of family A. Co-aggregation experimental assays revealed that the amyloid fibrils formed by the co-incubated peptide-analogues had a different morphology from that of the individual peptide-analogues’ fibrils. These data come to confirm the proposed homodimerization of family A proteins, as well as to suggest the segment that mainly guides the proteins’ A amyloid fibrillogenesis.
Overall, the results of this thesis confirmed the importance of the small sequence stretches with high amyloidogenic potential for guiding the folding of proteins into the amyloid state. These regions can be utilized for the future design of both targeted therapeutic approaches against amyloidosis and antimicrobial peptides. At the same time, the resulting data contributed to the enrichment of the list of amyloidogenic proteins in both humans and other organisms.
