Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution

Copper mediated amyloid-β binding to Transthyretin

Transthyretin (TTR), a homotetrameric protein that transports thyroxine and retinol both in plasma and in cerebrospinal (CSF) fluid provides a natural protective response against Alzheimer’s disease (AD), modulates amyloid-β (Aβ) deposition by direct interaction and co-localizes with Aβ in plaques. TTR levels are lower in the CSF of AD patients. Zn2+, Mn2+and Fe2+transform TTR into a protease able to cleave Aβ. To explain these activities, monomer dissociation or conformational changes have been suggested. Here, we report that when TTR crystals are exposed to copper or iron salts, the tetramer undergoes a significant conformational change that alters the dimer-dimer interface and rearranges residues implicated in TTR’s ability to neutralize Aβ. We also describe the conformational changes in TTR upon the binding of the various metal ions. Furthermore, using bio-layer interferometry (BLI) with immobilized Aβ(1–28), we observe the binding of TTR only in the presence of copper. Such Cu2+-dependent binding suggests a recognition mechanism whereby Cu2+modulates both the TTR conformation, induces a complementary Aβ structure and may participate in the interaction. Cu2+-soaked TTR crystals show a conformation different from that induced by Fe2+, and intriguingly, TTR crystals grown in presence of Aβ(1–28) show different positions for the copper sites from those grown its absence

CHARACTERIZATION OF β-2-MICROGLOBULIN PRE-AMYLOID OLIGOMERS AND THEIR ROLE IN AMYLOID INHIBITION

In dialysis patients, β-2 microglobulin (β2m) can aggregate and eventually form amyloid fibrils in a condition known as dialysis-related amyloidosis, which deleteriously affects joint, bone, and organ function, and eventually causes organ failure. To understand the early stages of the amyloid assembly process, we have employed a series of biophysical tools including chromatography, spectroscopy, and most especially, native electrospray ionization (ESI) together with ion mobility mass spectrometry (IM-MS) to study soluble pre-amyloid oligomeric species. We have also collaborated and integrated computational modeling to help better understand and rationalize the structural basis behind oligomerization. Recently, several small molecules have been identified as potential inhibitors of β2m amyloid formation in vitro. In two chapters of this dissertation, we investigate if these molecules are more broadly applicable inhibitors of β2m amyloid formation by studying their effect on Cu(II)-induced β2m amyloid formation and examine their inhibitory mechanisms. We found that three molecules (doxycycline, rifamycin SV, and epigallocatechin gallate) can inhibit β2m amyloid formation in vitroby causing the formation of amorphous, re-dissolvable aggregates. Rather than interfering with β2m amyloid formation at the monomer stage, we found that doxycycline and rifamycin SV exert their effect by binding to oligomeric species both in solution and in gas phase. Their binding results in a diversion of the expected Cu(II)-induced progression of oligomers toward a heterogeneous collection of oligomers, including trimers and pentamers, that ultimately matures into amorphous aggregates. EGCG is similar, generating a separate set of new oligomeric species that are ultimately off-pathway and distinctly non-fibrillar. Using IM-MS, we show doxycycline and rifamycin promote the compaction of the initially formed β2m dimer, which causes the formation of other off-pathway and amyloid-incompetent oligomers that are isomeric with amyloid-competent oligomers in some cases. Epigallocatechin gallate appears to deplete an important tetrameric conformer. Overall, our results suggest that doxycycline, rifamycin SV, and epigallocatechin gallate are general inhibitors of Cu(II)-induced β2m amyloid formation. Interestingly, the putative mechanism of their activity is different depending on how amyloid formation is initiated with β2m which underscores the complexity of how these structures assemble with different methods in vitro. With our ESI-IM-MS measurements, we revealed the presence of multiple conformers for the dimer, tetramer, and hexamer that precede the Cu(II)-induced amyloid assembly process, which is a brand new observation for this system. Experimental and computational results indicate that the predominant dimer is a Cu(II)-bound structure with an antiparallel side-by-side configuration. In contrast, tetramers exist in solution in both Cu(II)-bound and Cu(II)-free forms. Selective depletion of Cu(II)-bound species results in two primary conformers – one that is compact and another that is more expanded. Molecular modeling and molecular dynamics simulations identify models for these two tetrameric conformers with unique interactions and interfaces that enthalpically compensate for the loss of Cu(II). Unlike with other amyloid systems, conformational heterogeneity seems to be an essential aspect of Cu(II)-induced amyloid formation by β2m. Moreover, the Cu(II)-free models represent a new advance in our understanding of this critical event in Cu(II)-induced amyloid formation, laying a foundation for further mechanistic studies as well as development of new inhibition strategies. Finally, we end by presenting preliminary data on efforts that we have made in the lab to begin to better characterize the oligomeric conformers that we have detected. This is achieved primarily by performing tandem mass spectrometry experiments to study unfolding behavior/pathways, or by using solution phase labeling (i.e. deuterium) to enhance IM-MS

Monoaryl derivatives as transthyretin fibril formation inhibitors: Design, synthesis, biological evaluation and structural analysis

Transthyretin (TTR) is a ß-sheet-rich homotetrameric protein that transports thyroxine (T4) and retinol both in plasma and in cerebrospinal fluid. TTR also interacts with amyloid-β, playing a protective role in Alzheimer's disease. Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloids fibrillogenesis, and is responsible for extracellular deposition of amyloid fibrils. Small molecules, able to bind in T4 binding sites and stabilize the TTR tetramer, are interesting tools to treat and prevent systemic ATTR amyloidosis. We report here the synthesis, in vitro evaluation and three-dimensional crystallographic analyses of new monoaryl-derivatives in complex with TTR. Of the derivatives reported here, the best inhibitor of TTR fibrillogenesis, 1d, exhibits an activity similar to diflunisal

Protein Oligomerization

The chapter is a wide overview focused on protein oligomerization, a topic that definitely increased its importance in the last decades because of its connection to protein structure(s) and activity(ies). Protein oligomerization can occur naturally or can be induced artificially, either covalently or through different types of weak interactions. The main structural and functional features concerning these aspects are reported and discussed together with the aspects related to oligomers' stability and to the physiopathological consequences of protein oligomerization. Finally, some hints of possible industrial applications of protein oligomers are mentioned

The positive side of the Alzheimer's disease amyloid cross-interactions: The case of the Aβ 1-42 peptide with tau, TTR, CysC, and ApoA1

Alzheimer's disease (AD) represents a progressive amyloidogenic disorder whose advancement is widely recognized to be connected to amyloid-β peptides and Tau aggregation. However, several other processes likely contribute to the development of AD and some of them might be related to protein-protein interactions. Amyloid aggregates usually contain not only single type of amyloid protein, but also other type of proteins and this phenomenon can be rationally explained by the process of protein cross-seeding and co-assembly. Amyloid cross-interaction is ubiquitous in amyloid fibril formation and so a better knowledge of the amyloid interactome could help to further understand the mechanisms of amyloid related diseases. In this review, we discuss about the cross-interactions of amyloid-β peptides, and in particular Aβ1-42, with other amyloids, which have been presented either as integrated part of Aβ neurotoxicity process (such as Tau) or conversely with a preventive role in AD pathogenesis by directly binding to Aβ (such as transthyretin, cystatin C and apolipoprotein A1). Particularly, we will focus on all the possible therapeutic strategies aiming to rescue the Aβ toxicity by taking inspiration from these protein-protein interactions

Synthesis of novel vanillin derivatives: study of their antioxidant and potential neuroprotective properties.

Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a naturally-occurring phenolic compound, forming the main component of the bean and pod of vanilla orchids. It is widely used as a flavouring agent in food and drinks, and as a preservative in the cosmetic and pharmaceutical industries. In the past decades, several studies have reported on the antioxidant and protective effects of vanillin in several oxidative stress models, both in vitro and in vivo. The aim of this thesis was to synthesise novel vanillin derivatives with enhanced antioxidant properties and to study their potential neuroprotective activities in oxidative stress models in vitro. To achieve this aim, novel vanillin derivatives were synthesised through a reductive amination reaction, by reacting vanillin with a selection of amines. All the derivatives were characterized using 1H and 13C nuclear magnetic resonance and mass spectrometry. The vanillin derivatives were tested in several antioxidant assays with different mechanisms of action, in order to identify the functionalities that contributed to the antioxidant properties of this novel class of compounds. A structure-activity relationship (SAR) was therefore determined. The tetramer 4c turned out to be the most efficient antioxidant in all the assays. The latter compound consists of four vanillin moieties, together with a molecular structure that facilitates electron delocalisation for enhanced antioxidant activity. Selected based on their chemical structures and antioxidant properties, various vanillin derivatives were tested as potential multi-target-directed ligands (MTDLs), for use in the treatment of Alzheimer's disease (AD) - a multifactorial neurodegenerative disease. For this reason, the vanillin derivatives were tested for their ability to inhibit both the acetylcholinesterase (AChE) enzyme and the self-mediated A-beta(1-42) aggregation. The monomer 1f displayed the best inhibitory activities in both respects, with IC50 values at micro-M concentrations. In silico studies were performed in order to identify the molecular elements involved in the AChE inhibitory activities and to predict the ability of selected compounds to cross the blood-brain-barrier (BBB), which is of critical importance when targeting neurodegenerative diseases. Monomer 1f was predicted to be able to cross the BBB. Following this - and this time selected based on their antioxidant and AChE and amyloid inhibitory activities - another group of vanillin derivatives was then tested in oxidative stress models, by applying hydrogen peroxide or a mixture of rotenone/oligomycin A as stressors, in a neuroblastoma SH-SY5Y cell line. Vanillin derivatives showed cellular protective effects, for example by increasing cell viability and reducing reactive oxygen species (ROS) production. However, they were unable to protect the cells' DNA from oxidative damage. Again, compound 4c displayed the most efficient protective effects at micromolar concentrations. Finally, in order to study the mechanism behind the protective effects of 4c in SH-SY5Y cell line, research focused on its ability to activate the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway, which is known to be a predominant mediator of cellular antioxidant response. Since no Nrf2 was observed in the nucleus, this confirms that there must be an alternative mechanism for the antioxidant activity of 4c. Overall, compounds 1f and 4c showed promise for their further development with the potential to assist in the treatment of AD

Towards the elucidation of pathophysiology of amyloid conversion of globular proteins

Amyloidoses are a group of diseases caused by the conversion of soluble proteins into pathogenic ordered fibrillar aggregates. The mechanism driving in vivo the structural transformation of these proteins has not yet been clearly elucidated. My work has focused on two plasma proteins that make amyloid in vivo starting from precursors deeply different in terms of structure and function: transthyretin (TTR) and the apolipoprotein C-III variant, D25V (D25V apo C-III). Apo C-III is mostly synthesized by the liver and is a major component of HDL. We have described the first variant causing a genetic form of renal amyloidosis. In the work presented here, structural and functional characterization of the newly described D25V apo C-III variant was carried out together with the investigation of its aggregation mechanism, showing a modest loss of function and an increased tendency to aggregate in physiological buffer in the lipid-free state. TTR is mainly synthesized by the liver and the choroid plexus in the CSF and is the main transporter of thyroxine in the CSF and the secondary transporter in plasma. The mechanism of TTR fibrillogenesis has been investigated for decades. Our group has recently proposed a new pathway for TTR amyloidogenis mediated by selective tryptic cleavage, in alternative to the commonly accepted low pH induced aggregation mechanism. Further characterization of the mechanism, including the identification of the culprit protease responsible for proteolytic cleavage in vivo was carried out showing a correlation between TTR stability and susceptibility to proteolysis. The inhibitory activity of stabilisers and their effect on protein structure and dynamics were also studied using a combination of spectroscopic techniques including NMR. The identification of the enzyme responsible for cleavage in vivo, opens up a completely new scenario for understanding the mechanism and the history of the disease in vivo

Molecular Mechanism of Early Amyloid Self-Assembly Revealed by Computational Modeling

Protein misfolding followed by the formation of aggregates, is an early step in the cascade of conformational changes in a protein that underlie the development of several neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. Efforts aimed at understanding this process have produced little clarity and the mechanism remains elusive. Here, we demonstrate that the hairpin fold, a structure found in the early folding intermediates of amyloid b, induces morphological and stability changes in the aggregates of Aβ(14-23) peptide. We structurally characterized the interactions of monomer and hairpin using extended molecular dynamics (MD) simulations, which revealed a novel intercalated type complex. These finding suggest that folding patterns of amyloid proteins define the aggregation pathway. Computational analysis was then used to characterize the dimerization of full-length Aβ peptide and reveal their dynamic properties. Aβ dimers did not show β-sheet structures, as one may expect based on the known structures of Aβ fibrils, rather dimers are stabilized by hydrophobic interactions in the central hydrophobic regions. Comparison between Aβ40 and Aβ42 showed that overall, the dimers of both alloforms exhibit similar interaction strengths. However, the interaction patterns are significantly different. A novel aggregation pathway, able to describe aggregation at physiologically relevant concentrations, was elucidated when aggregation of amyloid proteins was performed in presence of surfaces. Computational analysis revealed that interaction of a monomer with the surface is accompanied by the structural transition of the monomer; which can then facilitate binding of another monomer and form a dimer. Compared to our previous data we observed an almost five-fold faster dimer formation. Further investigation of the surface-mediated aggregation revealed that lipid membranes promote aggregation of a-syn protein. MD simulations demonstrate that a-syn monomers change conformation upon interaction with the bilayers. On POPS, a-syn monomer protrudes from the surface. This conformation on POPS dramatically facilitates assembly of a dimer that remains stable over the entire simulation period. These findings are in line with experimental data. Overall, the studies described in this thesis provide the structural basis for the early stages of the misfolding and aggregation process of amyloid proteins