Molecular Mechanisms Of Aggregation Research Articles

Tunable molecular packing modes via H- or J-aggregates in the supramolecular helical nanostructures from an achiral C3 symmetric molecule.

The self-assembly of a C3-symmetric molecule benzene-1,3,5-tricarboxylate substituted with methyl cinnamate (BTECM) has been investigated by a reprecipitation method in H2O and cetyltrimethylammonium bromide (CTAB) aqueous solution, respectively. The nanostructures and characteristics of the assemblies were monitored by UV-Vis spectroscopy, fluorescence (FL) spectroscopy, circular dichroism (CD) spectroscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). It was found that helical nanostructures were successfully assembled from the achiral C3 molecule BTECM. More importantly, the helices aggregated via different packing modes in H2O and CTAB aqueous solution. In H2O, the nanostructures underwent a process of particles, fibers and helices via H-type aggregate upon aging. In the case of CTAB aqueous solution (1.2 mM), the helices were translated from particles and the molecules were inclined to aggregate via the J-type mode. In addition, the aggregation process could be accelerated by raising the temperature proved by UV-Vis spectra. A molecular aggregation mechanism was proposed based on the experimental results.

Systematic generation and imaging of tandem repeats reveal base-pairing properties that promote RNA aggregation

A common pathological feature of RNAs containing expanded repeat sequences is their propensity to aggregate in cells. While some repeat RNA aggregates have been shown to cause toxicity by sequestering RNA-binding proteins, the molecular mechanism of aggregation remains unclear. Here, we devised an efficient method to generate long tandem repeat DNAs de novo and applied it to systematically determine the sequence features underlying RNA aggregation. Live-cell imaging of repeat RNAs indicated that aggregation was promoted by multivalent RNA-RNA interactions via either canonical or noncanonical base pairs. While multiple runs of two consecutive base pairs were sufficient, longer runs of base pairs such as those formed by GGGGCC hexanucleotide repeats further enhanced aggregation. In summary, our study provides a unifying model for the molecular basis of repeat RNA aggregation and a generalizable approach for identifying the sequence and structural determinants underlying the distinct properties of repeat DNAs and RNAs.

Toxic Misfolded Transthyretin Oligomers with Different Molecular Conformations Formed through Distinct Oligomerization Pathways.

Protein aggregation is initiated by structural changes from native polypeptides to cytotoxic oligomers, which form cross-β structured amyloid. Identification and characterization of oligomeric intermediates are critically important for understanding not only the molecular mechanism of aggregation but also the cytotoxic nature of amyloid oligomers. Preparation of misfolded oligomers for structural characterization is, however, challenging because of their transient, heterogeneous nature. Here, we report two distinct misfolded transthyretin (TTR) oligomers formed through different oligomerization pathways. A pathogenic TTR variant with a strong aggregation propensity (L55P) was used to prepare misfolded oligomers at physiological pH. Our mechanistic studies showed that the full-length TTR initially forms small oligomers, which self-assemble into short protofibrils at later stages. Enzymatic cleavage of the CD loop was also used to induce the formation of N-terminally truncated oligomers, which was detected in ex vivo cardiac TTR aggregates extracted from the tissues of patients. Structural characterization of the oligomers using solid-state nuclear magnetic resonance and circular dichroism revealed that the two TTR misfolded oligomers have distinct molecular conformations. In addition, the proteolytically cleaved TTR oligomers exhibit a higher surface hydrophobicity, suggesting the presence of distinct oligomerization pathways for TTR oligomer formation. Cytotoxicity assays also revealed that the cytotoxicity of cleaved oligomers is stronger than that of the full-length TTR oligomers, indicating that hydrophobicity might be an important property of toxic oligomers. These comparative biophysical analyses suggest that the toxic cleaved TTR oligomers formed through a different misfoling pathway may adopt distinct structural features that produce higher surface hydrophobicity, leading to the stronger cytotoxic activities.

Implementing Complementary Approaches to Shape the Mechanism of α-Synuclein Oligomerization as a Model of Amyloid Aggregation.

The aggregation of proteins into amyloid fibers is linked to more than forty still incurable cellular and neurodegenerative diseases such as Parkinson’s disease (PD), multiple system atrophy, Alzheimer’s disease and type 2 diabetes, among others. The process of amyloid formation is a main feature of cell degeneration and disease pathogenesis. Despite being methodologically challenging, a complete understanding of the molecular mechanism of aggregation, especially in the early stages, is essential to find new biological targets for innovative therapies. Here, we reviewed selected examples on α-syn showing how complementary approaches, which employ different biophysical techniques and models, can better deal with a comprehensive study of amyloid aggregation. In addition to the monomer aggregation and conformational transition hypothesis, we reported new emerging theories regarding the self-aggregation of α-syn, such as the alpha-helix rich tetramer hypothesis, whose destabilization induce monomer aggregation; and the liquid-liquid phase separation hypothesis, which considers a phase separation of α-syn into liquid droplets as a primary event towards the evolution to aggregates. The final aim of this review is to show how multimodal methodologies provide a complete portrait of α-syn oligomerization and can be successfully extended to other protein aggregation diseases.

Folding Steps in the Fibrillation of Functional Amyloid: Denaturant Sensitivity Reveals Common Features in Nucleation and Elongation

Functional bacterial amyloids (FuBA) are intrinsically disordered proteins (IDPs) which rapidly and efficiently aggregate, forming extremely stable fibrils. The conversion from IDP to amyloid is evolutionarily optimized and likely couples folding to association. Many FuBA contain several imperfect repeat sequences which contribute to the stability of mature FuBA fibrils. Aggregation can be considered an intermolecular extension of the process of intramolecular protein folding which has traditionally been studied using chemical denaturants. Here we employ denaturants to investigate folding steps during fibrillation of CsgA and FapC. We quantify protein compactification (i.e. the extent of burial of otherwise exposed surface area upon association of proteins) during different stages of fibrillation based on the dependence of fibrillation rate constants on the denaturant concentration (m-values) determined from fibrillation curves. For both proteins, urea mainly affects nucleation and elongation (not fragmentation), consistent with the fact that these steps involve both intra- and intermolecular association. The two steps have similar m-values, indicating that activation steps in nucleation and elongation involve the same level of folding. Surprisingly, deletion of two or three repeats from FapC leads to larger m-values (i.e. higher compactification) during the activation step of fibril growth. This observation is extended by SAXS analysis of the fibrils which indicates that weakening of the amyloidogenic core caused by repeat deletions causes a larger portion of normally unstructured regions of the protein to be included into the amyloid backbone. We conclude that the sensitivity of fibrillation to denaturants can provide useful insight into molecular mechanisms of aggregation.

The curvature of gold nanoparticles influences the exposure of amyloid-β and modulates its aggregation process

Gold nanoparticles (GNP) are tunable nanomaterials that can be used to develop rational therapeutic inhibitors against the formation of pathological aggregates of proteins. In the case of the pathological aggregation of the amyloid-β protein (Aβ), the shape of the GNP can slow down or accelerate its aggregation kinetics. However, there is a lack of elementary knowledge about how the curvature of GNP alters the interaction with the Aβ peptide and how this interaction modifies key molecular steps of fibril formation. In this study, we analysed the effect of flat gold nanoprisms (GNPr) and curved gold nanospheres (GNS) on in vitro Aβ42 fibril formation kinetics by using the thioflavin-based kinetic assay and global fitting analysis, with several models of aggregation. Whereas GNPr accelerate the aggregation process and maintain the molecular mechanism of aggregation, GNS slow down this process and modify the molecular mechanism to one of fragmentation/secondary nucleation, with respect to controls. These results can be explained by a differential interaction between the Aβ peptide and GNP observed by Raman spectroscopy. While flat GNPr expose key hydrophobic residues involved in the Aβ peptide aggregation, curved GNS hide these residues from the solvent. Thus, this study provides mechanistic insights to improve the rational design of GNP nanomaterials for biomedical applications in the field of amyloid-related aggregation.

Hybrid resolution molecular dynamics simulations of amyloid proteins interacting with membranes.

A broad range of human diseases, including Alzheimer's and Parkinson's diseases, arise from or have as key players intrinsically disordered proteins. The aggregation of these amyloid proteins into fibrillar aggregates are the key events of such diseases. Characterizing the conformation dynamics of the proteins involved is crucial for understanding the molecular mechanisms of aggregation, which in turn is important for drug development efforts against these diseases. Computational approaches have provided extensive detail about some steps of the aggregation process, however the biologically relevant elements responsible for the aggregation and or aggregation propagation have not been fully characterized. Here we describe a hybrid resolution molecular dynamics simulation method that can be employed to investigate the interaction of amyloid proteins with lipid membranes, shown to dramatically accelerate the aggregation propensity of amyloid proteins. The hybrid resolution method enables routine and accurate simulation of multi-protein and complex membrane systems, mimicking biologically relevant lipid membranes, on microsecond time scales. The hybrid resolution method was applied to computer modeling of the interactions of α -synuclein protein with a mixed lipid bilayer.

Aggregation of Aβ40/42 chains in the presence of cyclic neuropeptides investigated by molecular dynamics simulations.

Alzheimer’s disease is associated with the formation of toxic aggregates of amyloid beta (Aβ) peptides. Despite tremendous efforts, our understanding of the molecular mechanisms of aggregation, as well as cofactors that might influence it, remains incomplete. The small cyclic neuropeptide somatostatin-14 (SST14) was recently found to be the most selectively enriched protein in human frontal lobe extracts that binds Aβ42 aggregates. Furthermore, SST14’s presence was also found to promote the formation of toxic Aβ42 oligomers in vitro. In order to elucidate how SST14 influences the onset of Aβ oligomerization, we performed all-atom molecular dynamics simulations of model mixtures of Aβ42 or Aβ40 peptides with SST14 molecules and analyzed the structure and dynamics of early-stage aggregates. For comparison we also analyzed the aggregation of Aβ42 in the presence of arginine vasopressin (AVP), a different cyclic neuropeptide. We observed the formation of self-assembled aggregates containing the Aβ chains and small cyclic peptides in all mixtures of Aβ42–SST14, Aβ42–AVP, and Aβ40–SST14. The Aβ42–SST14 mixtures were found to develop compact, dynamically stable, but small aggregates with the highest exposure of hydrophobic residues to the solvent. Differences in the morphology and dynamics of aggregates that comprise SST14 or AVP appear to reflect distinct (1) regions of the Aβ chains they interact with; (2) propensities to engage in hydrogen bonds with Aβ peptides; and (3) solvent exposures of hydrophilic and hydrophobic groups. The presence of SST14 was found to impede aggregation in the Aβ42–SST14 system despite a high hydrophobicity, producing a stronger “sticky surface” effect in the aggregates at the onset of Aβ42–SST14 oligomerization.

Phase Separation and Neurodegenerative Diseases: A Disturbance in the Force.

Protein aggregation is the main hallmark of neurodegenerative diseases. Many proteins found in pathological inclusions are known to undergo liquid-liquid phase separation, a reversible process of molecular self-assembly. Emerging evidence supports the hypothesis that aberrant phase separation behavior may serve as a trigger of protein aggregation in neurodegeneration, and efforts to understand and control the underlying mechanisms are underway. Here, we review similarities and differences among four main proteins, α-synuclein, FUS, tau, and TDP-43, which are found aggregated in different diseases and were independently shown to phase separate. We discuss future directions in the field that will help shed light on the molecular mechanisms of aggregation and neurodegeneration.

Computational studies of protein aggregation mediated by amyloid: Fibril elongation and secondary nucleation.

Amyloid formation by proteins and peptides is the hallmark of many diseases. Growing evidence suggests that oligomeric species arising during aggregation are toxic, but the molecular mechanism of aggregation and oligomer generation remains unclear. Recent discoveries that amyloid fibrils can convert soluble proteins into oligomeric nuclei to facilitate aggregation highlight the role played by fibrils in protein aggregation. We review here computational studies conducted to elucidate the molecular mechanism of two fibril-dependent processes during protein aggregation, namely, secondary nucleation and fibril elongation. Secondary nucleation occurs on the lateral surface of a fibril to generate nuclei while fibril elongation, through addition of proteins to the ends of fibrils increases the lateral surface of the fibril. We summarize the molecular insights into each process unraveled by computational methods at levels ranging from coarse-grained to atomic and discuss the connection between these insights and experimental observations. The computational challenges faced by these studies and their solutions are also discussed. Finally, we propose possible computational studies that could shed light on the mechanistic aspects of secondary nucleation and fibril elongation that have been unaddressed.

MISFOLDING AND EARLY AGGREGATION OF AMYLOID β42 PROTEIN: THE CRITICAL ROLE OF MEMBRANES

Even with growing evidence for the involvement of Aβ oligomers in the development of Alzheimer's disease (AD), very limited knowledge exists regarding the molecular mechanisms behind the protein aggregation processes for Aβ. The amyloid cascade hypothesis posits that the onset of diseases due to protein aggregation involves the spontaneous assembly of an amyloidogenic polypeptide. However, there is a serious complication with translating current knowledge on amyloid aggregation in vitro to understanding the aggregation process in vivo. We combined AFM imaging and high-power computational approaches to elucidate a molecular mechanism of aggregation of Aβ42 on membranes. We have discovered that interaction of Aβ42 with membranes allows for Aβ42 to assemble in oligomers at the physiological concentration range. We were able to directly visualize a spontaneous assembly of Aβ42 aggregates on the membrane bilayers. Importantly, this aggregation process is dynamic, so the assembled aggregates can dissociate from the surface into the bulk solution and play roles of seeds for aggregation in the bulk solution, or start a neurotoxic effect such as phosphorylation of the tau protein to initiate its misfolding and aggregation. To elucidate a molecular mechanism underlying the on-surface aggregation process, we applied computational modeling (all-atom Molecular Dynamic simulations). The simulations demonstrate that the interaction with the membrane surface dramatically changes the conformation of Aβ42 protein allowing for the formation of long aggregation–prone β-sheet structures never observed for Aβ42 protein in solution. We posit that on-membrane aggregation is the mechanism by which neurotoxic amyloid aggregates are assembled under physiological conditions. We propose a model in which amyloid aggregation is linked with elevated synthesis of amyloid proteins. A change in membrane properties leading to an increase in affinity of amyloid proteins to the membrane surface facilitates the assembly of stable oligomers. The proposed model is a significant departure from the current model as it directs the development of treatments and preventions towards approaches that control the cell membranes composition to prevent the on-surface aggregation process.

Dynamic formation of cellular aggregates of chondrocytes and mesenchymal stem cells in spinner flask.

ObjectivesCellular aggregates are readily applicable in cell‐based therapy. The effects of agitation and inoculation density on the aggregation of cells in spinner flask and the molecular mechanism of aggregation were investigated.Materials and methodsThe aggregation kinetics of cells in spinner flask was evaluated with bovine articular chondrocytes (bACs), rabbit bone marrow‐derived mesenchymal stem cells (rMSCs) and their mixture. The morphology of cellular aggregates was studied with scanning electron microscopy and gene expression of cell adhesion‐related molecules was analysed.ResultsIt was shown that suspension culture in spinner flask induced the aggregation of bACs and rMSCs. Both cells exhibited increased aggregation rate and aggregate size with decreasing agitation rate and increasing cell inoculation density. Additionally, aggregate size increased with extended culture time. By analysing gene expression of integrin β1 and cadherin, it was indicated that these molecules were potentially involved in the aggregation process of bACs and rMSCs, respectively. Aggregates composed of both bACs and rMSCs were also prepared, showing rMSCs in the core and bACs in the periphery.ConclusionsCellular aggregates were prepared in dynamic suspension culture using spinner flask, the key parameters to the aggregation process were identified, and the molecular mechanism of aggregation was revealed. This would lay a solid foundation for the large‐scale production of cellular aggregates for cell‐based therapy, such as cartilage regeneration.

Structural insights into the aggregation mechanism of huntingtin exon 1 protein fragment with different polyQ-lengths.

Huntington disease is a neurodegenerative disorder caused by the expansion of polyglutamine (polyQ) at the N-terminal of the huntingtin exon 1 protein. The detailed structure and the mechanism behind this aggregation remain unclear and it is assumed that the polyQ undergoes a conformational transition to the β-sheet structure when it aggregates. Investigating the misfolding of polyQ facilitates the determination of the molecular mechanism of aggregation and can potentially help in developing a novel approach to inhibit polyQ aggregation. Moreover, the flanking sequences of the polyQ region play a vital role in structural changes and the aggregation mechanism. We performed all-atom molecular dynamics simulations to gain structural insights into the aggregation mechanism using eight different models with glutamine repeat lengths Q27 , Q27 P11 , Q34 , Q35 , Q36 , Q40 , Q50 , and Q50 P11 . In the models without flanking polyPs, we noticed that the transformation of a random coil to β-sheet occurs when the number of Q increases. We also found that the flanking polyPs prevent aggregation by decreasing the probability of forming a β-sheet structure. When polyQ length increases, the 17 N-terminal flanking residues are more likely to adopt a β-sheet conformation from α-helix and coil. From our simulations, we suggest that at least 34 glutamines are required for initiating aggregation and 40 residues length is critical for the aggregation of huntingtin exon 1 protein for disease onset. This study provides structural insights into misfolding and the role of flanking sequences in huntingtin aggregation which will further help in developing therapeutic strategies for Huntington's disease.

Investigation of molecular aggregation mechanism of glipizide/cyclodextrin complexation by combined experimental and molecular modeling approaches

Cyclodextrin complexation is a wise strategy to enhance aqueous solubility of water-insoluble drugs. However, the aggregation mechanism of drug-cyclodextrin complexes is still unclear. This research aimed to investigate the molecular aggregation mechanism of glipizide/cyclodextrin complexation by the combination of experimental and modeling methods. Binding free energies between glipizide and cyclodextrins from modeling calculations were higher than those by the phase solubility diagram method. Both experimental and modeling results showed that methylated-β-cyclodextrin exhibited the best solubilizing capability to glipizide. Size-measurement results confirmed the aggregation between glipizide and all four cyclodextrins in high concentrations. Glipizide/γ-cyclodextrin and glipizide/β-cyclodextrin complexes showed stronger aggregation trend than HP-β-cyclodextrin and methylated-β-cyclodextrin. The substituted groups in the rim of HP-β-cyclodextrin and methylated-β-cyclodextrin lead to weak aggregation. This research provided us a clear molecular mechanism of glipizide/cyclodextrin complexation and aggregation. This research will also benefit the formulation development of cyclodextrin solubilization.

\u03b1-Synuclein interacts directly but reversibly with psychosine: implications for \u03b1-synucleinopathies

Aggregation of α-synuclein, the hallmark of α-synucleinopathies such as Parkinson’s disease, occurs in various glycosphingolipidoses. Although α-synuclein aggregation correlates with deficiencies in the lysosomal degradation of glycosphingolipids (GSL), the mechanism(s) involved in this aggregation remains unclear. We previously described the aggregation of α-synuclein in Krabbe’s disease (KD), a neurodegenerative glycosphingolipidosis caused by lysosomal deficiency of galactosyl-ceramidase (GALC) and the accumulation of the GSL psychosine. Here, we used a multi-pronged approach including genetic, biophysical and biochemical techniques to determine the pathogenic contribution, reversibility, and molecular mechanism of aggregation of α-synuclein in KD. While genetic knock-out of α-synuclein reduces, but does not completely prevent, neurological signs in a mouse model of KD, genetic correction of GALC deficiency completely prevents α-synuclein aggregation. We show that psychosine forms hydrophilic clusters and binds the C-terminus of α-synuclein through its amino group and sugar moiety, suggesting that psychosine promotes an open/aggregation-prone conformation of α-synuclein. Dopamine and carbidopa reverse the structural changes of psychosine by mediating a closed/aggregation-resistant conformation of α-synuclein. Our results underscore the therapeutic potential of lysosomal correction and small molecules to reduce neuronal burden in α-synucleinopathies, and provide a mechanistic understanding of α-synuclein aggregation in glycosphingolipidoses.

On the role of sidechain size and charge in the aggregation of Aβ42 with familial mutations

The aggregation of the amyloid-β (Aβ) peptide is linked to the pathogenesis of Alzheimer's disease (AD). In particular, some point mutations within Aβ are associated with early-onset familial Alzheimer's disease. Here we set out to explore how the physical properties of the altered side chains, including their sizes and charges, affect the molecular mechanisms of aggregation. We focus on Aβ42 with familial mutations-A21G (Flemish), E22K (Italian), E22G (Arctic), E22Q (Dutch), and D23N (Iowa)-which lead to similar or identical pathology with sporadic AD or severe cerebral amyloid angiopathy. Through global kinetic analysis, we find that for the E22K, E22G, E22Q, and D23N mutations, the acceleration of the overall aggregation originates primarily from the modulation of the nucleation processes, in particular secondary nucleation on the surface of existing fibrils, whereas the elongation process is not significantly affected. Remarkably, the D23 position appears to be responsible for most of the charge effects during nucleation, while the size of the side chain at the E22 position plays a more significant role than its charge. Thus, we have developed a kinetic approach to determine the nature and the magnitude of the contribution of specific residues to the rate of individual steps of the aggregation reaction, through targeted mutations and variations in ionic strength. This strategy can help rationalize the effect of some disease-related mutations as well as yield insights into the mechanism of aggregation and the transition states of the wild-type protein.

Evaluating Polyglutamine Peptide Monomers and Dimers with Enhanced Sampling MD Simulations

Huntington's disease is a neurodegenerative disease characterized by mutations causing polyglutamine (polyQ) repeats in proteins. Mutated proteins misfold, aggregate, and form amyloid-like fibrils in the neuron. Experimental techniques such as resonance Raman, circular dichroism, and ssNMR are used to analyze properties of polyQ systems, but lack the ability to monitor the molecular aggregation mechanism. In this work, polyQ peptide monomers and dimers are studied using molecular dynamics (MD) methods in order to better understand early stages of aggregation. We have characterized the monomeric conformational ensemble of D2Q10K2 peptides in aqueous and protein-globule environments. We also investigate dimerization properties of these peptides. Adaptive biasing force paired with MD is used to evaluate the dimerization free energies and conformations of D2Q10K2 peptides. Classical MD is used to simulate the favorable dimeric conformations in equilibrium, as to better understand the interactions and dynamics of these structures. These results will be presented and discussed.

B06 Exploring The Role Of Phosphorylation In Huntingtin Aggregation Dynamics

Background Huntington’s disease (HD) is characterised by misfolding and aggregation of mutant huntingtin (HTT), a dynamic process that starts with the association of misfolded HTT monomers into soluble oligomeric structures. Current evidence suggests that dimers and oligomers are the most toxic HTT species while large aggregates may in fact be neuroprotective. To better understand HTT toxicity it is essential to understand the molecular mechanisms of aggregation. Aims Our goal was to elucidate the role of HTT N-terminal phosphorylation in the aggregation and toxicity of mutant HTT. Methods/techniques Bimolecular fluorescence complementation (BiFC) was employed to visualise soluble and insoluble HTT species. We used this model to study the behaviour of HTT phosphomimic and phosphoresistant mutants in living cells. Moreover, we tested several compounds that modulate phosphorylation in Drosophila expressing HTT exon 1 (HTT93Q) under the control of the GAL4/UAS system. Pan-neuronal expression of HTT93Q by elavGAL4 gives a variety of disease-relevant phenotypes, including degeneration of photoreceptor neurons. We compared the neurodegeneration of treated or non-treated HD flies using the light microscopy-based pseudopupil assay. Results/outcome When phosphomimic mutations were present in HTT103Q BiFC constructs, the generation of inclusion bodies was completely abolished. Phosphomimic and Phosphoresistant mutants had varied effects on HTT toxicity, which were not associated with the levels of oligomers or inclusion bodies. A CDC25 phosphatase inhibitor decreased aggregation in cells expressing HTT103Q and increased neurodegeneration in HTT93Q flies. Notably, a specific protein phosphatase 2A (PP2A) inhibitor completely reversed HTT93Q neurotoxicity in Drosophila. Conclusions The phosphorylation state of HTT N-terminal region plays a role in aggregation and toxicity of the protein, and this can be pharmacologically modulated by targeting cellular phosphatases.

Aggregation States Driven by Dipole Interactions Regulates Action Mechanism of Polyene Antibiotics

Due to its clinical relevance, the action mechanism of the polyene antibiotic Amphotericin B (AmB) has been studied extensively using both experimental (Venegas-Cotero et al 2003, Kasai et al. 2008) and theoretical (Baginski et al. 2005, Czub et al. 2007) approaches. AmB is currently the drug of choice for treating advanced systemic fungal infections in spite of having a strong collateral toxicity. There is evidence showing that AmB acts at the cellular membrane. However, AmB aggregation could occur in the aqueous solution previous to absorption into the membrane. These aggregation states have been proposed as one of the molecular factors responsible for its action mechanism (Bolard 1991, Szlinder-Richert 2001). The molecular details of this process, in terms of spatial configuration and interaction energy, are poorly understood.In this work, the molecular aggregation mechanism, as a function of AmB concentration in water, was studied through molecular dynamics. Hydrophobic forces, hydrogen bonding and dipole-dipole interactions were characterized. At low concentration not all possible dimer configurations were observed. After running a Potential of Mean Force analysis, one dimer configuration was identified as the most energetically favorable. In this, both molecules are interacting head to tail in an antiparallel fashion. When increasing concentration, the drug still aggregates quickly (within 50 ns), forming higher aggregates. In all aggregation states found, dipole-dipole interactions were identified as the main interacting force. In addition, since AmB is believed to absorb into the membrane, Free energy of solvation in low dielectric media, and the partition coefficient as a function of aggregation state, were calculated. Our results show that AmB aggregates before membrane absorption.

Conformational Diseases: Structural Studies of Aggregation of Polyglutamine Proteins

Protein misfolding and aggregation into insoluble amyloid deposits are often associated with neurodegenerative disorders. In particular, the polyglutamine (polyQ) diseases are inherited disorders triggered by the expansion of the polyQ tract over its physiological length in the involved protein. The molecular mechanism of aggregation from the native protein into amyloids involves several steps including protein misfolding, aggregation into oligomers, which seems to be the most toxic species, and, finally rearrangements into mature fibrils. In the present contribution, we review studies, integrating computational and experimental approaches, of polyQ proteins, as well as of the details of the complicate aggregation mechanisms in which aberrant form of polyQ proteins are involved. These aspects are of crucial relevance for a complete understanding of the onset of polyQ conformational diseases and can also shed light on putative therapeutic targets and future development of aggregation inhibitors.