Aggregation of proteins into amyloid deposits is a common feature among dozens of diseases. Two such diseases that feature amyloid deposits are Alzheimer's disease (AD) and type 2 diabetes (T2D). AD toxicity has been associated with the aggregation and accumulation of the amyloid β-peptide (Aβ); Aβ exerts its toxic effects through interactions with neuronal cell membranes. A characteristic feature of T2D is the deposition of the islet amyloid polypeptide (IAPP) in the pancreatic islets of Langerhans. It is currently unknown if IAPP aggregation is a cause or consequence of T2D, but it does lead to β-cell dysfunction and death, exacerbating the effects of diabetes. Characterizing the fundamental interactions between both Aβ and IAPP with lipid membranes and in solution will give greater insight into mechanisms of toxicity exhibited by amyloid proteins. In this work, molecular dynamics (MD) simulations were used to study the secondary, tertiary, and quatnary structure of Aβ and IAPP, in addition to peptide-membrane interactions and membrane perturbation as independently caused by both peptides. Studies were conducted to address the following questions: (1) what influence do solution conditions and oxidation state have on monomeric Aβ] (2) how and in what way does monomeric Aβ interact with model lipid membranes and what role does sequence play on these peptide-membrane interactions; (3) can MD simulations be utilized to understand Aβ tetramer formation, rearrangement, and tetramer-membrane interactions; (4) how does IAP interact with model membranes and how does that vary from non-toxic (rat) IAPP peptide-membrane interactions. These studies led to conclusions that showed variance in lipid affinity and degree of perturbation as based on peptide sequence, in addition to insight into the type of perturbation caused to membranes by these amyloid peptides. Understanding the differences in peptide-membrane interactions of amyloidogenic and non-amyloidogenic (rat) peptides gave insight into the overall mechanism of amyloidogenicity, leading to the detection of specific amino acids essential in peptide-membrane perturbation. These residues can then be targeted for novel therapeutic design to attenuate the perturbation and potential cell death as caused by these peptides. / Ph. D.

<p>This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR.</p><p>The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes.</p><p>mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system.</p><p>Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.</p>

A combined attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR)-atomic force microscopy (AFM) platform was used to visualize and characterize membranolytic protein- and peptide-membrane interactions, allowing spectroscopic details to be correlated with structural features. Modifications to a previous combined platform permitted IR results for physiologically-relevant protein or peptide concentrations as well as provided nanometer-resolution height data for AFM. This combination provides greater insight than individual techniques alone. The interactions of hemolytic sticholysin proteins on a model red blood cell membrane showed evidence of conformational changes associated with a membrane-induced organization. In addition, the examination of a de novo cationic antimicrobial peptide on a model bacterial membrane showed that the peptide adopted a helical structure upon interaction with the membrane, and also provided evidence of membrane disruption and peptide aggregation. These results demonstrate that ATR-FTIR-AFM can be a powerful tool for understanding protein- and peptide-membrane interactions.

A combined attenuated total reflection-Fourier-transform infrared spectroscopy (ATR-FTIR)-atomic force microscopy (AFM) platform was used to visualize and characterize membranolytic protein- and peptide-membrane interactions, allowing spectroscopic details to be correlated with structural features. Modifications to a previous combined platform permitted IR results for physiologically-relevant protein or peptide concentrations as well as provided nanometer-resolution height data for AFM. This combination provides greater insight than individual techniques alone. The interactions of hemolytic sticholysin proteins on a model red blood cell membrane showed evidence of conformational changes associated with a membrane-induced organization. In addition, the examination of a de novo cationic antimicrobial peptide on a model bacterial membrane showed that the peptide adopted a helical structure upon interaction with the membrane, and also provided evidence of membrane disruption and peptide aggregation. These results demonstrate that ATR-FTIR-AFM can be a powerful tool for understanding protein- and peptide-membrane interactions.

This thesis focuses on peptides derived from the Prion, Doppel and Influenza haemagglutinin proteins in the context of bilayer interactions with model membranes and live cells. The studies involve spectroscopic techniques like fluorescence, fluorescence correlation spectroscopy (FCS), circular and linear dichroism (CD and LD), confocal fluorescence microscopy and NMR. The peptides derived from the Prion and Doppel proteins combined with their subsequent nuclear localization-like sequences, makes them resemble cell-penetrating peptides (CPPs). mPrPp(1-28), corresponding to the first 28 amino acids of the mouse PrP, was shown to translocate across cell membranes, concomitantly causing cell toxicity. Its bovine counterpart bPrPp(1-30) was demonstrated to enter live cells, with and without cargo, mainly via macropinocytosis. The mPrPp(23-50) peptide sequence overlaps with mPrPp(1-28) sharing the KKRPKP sequence believed to encompass the driving force behind translocation. mPrPp(23-50) was however found unable to cross over cell membranes and had virtually no perturbing effects on membranes. mDplp(1-30), corresponding of the first 30 N-terminal amino acids of the Doppel protein, was demonstrated to be almost as membrane perturbing as melittin. NMR experiments in bicelles implied a transmembrane configuration of its alpha-helix, which was corroborated by LD in vesicle bilayers. The positioning of the induced alpha-helix in transportan was found to be more parallel to the bilayer surface in the same model system. Positioning of the native Influenza derived fusion peptide in bilayers showed no pH dependence. The glutamic acid enriched variant however, changed its insertion angle from 70 deg to a magic angle alignment relative the membrane normal upon a pH drop from 7.4 to 5.0. Concomitantly, the alpha-helical content dramatically rose from 18% to 52% in partly anionic membranes, while the native peptide’s helicity increased only from 39% to 44% in the same conditions.

Orientador: José Roberto Ruggiero / Banca: Pedro Geraldo Pascutti / Banca: José Maria Pires / Banca: Alexandre Suman de Araújo / Banca: Sabrina Thais Broggio Costa / Resumo: Peptídeos antimicrobianos são moléculas biologicamente ativas que, geralmente, tem as membranas fosfolipídicas como alvo primário. Resultados de diferentes técnicas experimentais têm sugerido que esses peptídeos permeabilizam as membranas pela formação de poros. Parte dos peptídeos caracterizados apresentam especificidade de disrupção para membranas de bactérias, em detrimento das membranas dos hospedeiros. Essa característica tem atraído a atenção da comunidade científica internacional, porque indica que estas moléculas podem ser modelos para o desenvolvimento de novos antibióticos, portanto o entendimento do mecanismo de ação, ou seja, do mecanismo de formação de poro, tem extrema importância. Simulações por Dinâmica Molecular foram produzidas para investigarmos o impacto que peptídeos antimicrobianos da família Mastoparano tem sobre membranas lipídicas modelo. Dois cenários foram explorados: (i) de baixa concentração peptídeo/lipídeo, P/L=1/128, que consistia de simulações fine-grained das interações de um peptídeo com uma bicamada pura de 128 lipídeos aniônicos (POPG) ou zwiteriônicos (POPC); (ii) de alta concentração, P/L=1/21, que abordava as interações de seis peptídeos com uma bicamada mista de 128 lipídeos POPC/POPG (1/1) usando uma modelagem coarse-grained. Tomando o peptídeo MP1 como caso paradigmático, verificamos que em baixo P/L é possível sugerir que sua característica seletiva surge da capacidade de coordenar e perturbar maior número de lipídeos em membrana aniônica comparada à neutra. Essa capacidade fica acentuada nas simulações com membrana mista, onde a atração dos lipídeos aniônicos pelos peptídeos catiônicos guiou a separação local e a formação de domínios de lipídeos aniônicos, o que facilitou o afinamento local da membrana e a formação de poro transmembrânico. Esses achados ajudam a explicar como peptídeos / Abstract: Antimicrobial peptides are biologically active molecules that, usually, have the phospholipid membranes as a primary target. Results from different experimental techniques have suggested these peptides permeabilize membranes by the pore formation. Part of the characterized peptides have specificity of disruption for bacterial membranes, instead of host membrane. This feature has attracted the attention of the international scientific community, because it indicates that these molecules can be models for the development of novel antibiotics, so understanding the mechanism of action, ie, the mechanism of pore formation, is extremely important. Molecular dynamics simulations were performed to investigate the impact of antimicrobial peptides from the Mastoparano family have on model lipid membranes. Two scenarios were explored: (i) of low peptide/lipid concentration, P/L=1/128, which consisted of fine-grained simulations of the interactions of a peptide with a pure bilayer of 128 anionic (POPG) or zwitterionic (POPC) lipids; (ii) of high concentration, P/L=1/21, which addressed the interactions of six peptides with a mixed bilayer of 128 POPC/POPG (1/1) lipids, using a coarse-grained modeling. Taking the MP1 peptide as a paradigmatic case, we found that in low P/L is possible to suggest that its selective feature arises of its ability to coordinate and disturb large number of lipids in the anionic membrane compared to neutral one. This ability is accentuated in simulations with mixed membrane, where the attraction of the anionic lipids by the cationic peptides led to the local segregation and formation of POPG lipid domains, which facilitated the local thinning of the membrane and the formation of transmembrane pore. These findings help to explain how short peptides, such as MP1, are able of forming pores in a membrane whose thickness is larger than the length of the peptide / Doutor