Estudo da interação entre péptidos e membranas celulares por dinâmica molecular e ensaios experimentais : o caso dos péptidos com atividade membranar

Dissertação de mestrado em Biofísica e BionanossistemasA interação entre péptidos e membranas tem um papel fundamental num elevado número de processos, tais como nas funções das proteínas de membrana, na atividade antimicrobiana, nas infeções virais, bem como em processos que originam doenças graves como as neuro-degenerativas Alzheimer e Parkinson. Péptidos penetrantes de células (do Inglês “Cell-penetrating peptides” - CPPs) e péptidos antimicrobianos (do Inglês “Antimicrobial peptides” - AMPs) são geralmente definidos como pequenos péptidos catiónicos com a propriedade de interagirem com membranas lipídicas, num processo dirigido pelas suas propriedades eletrostáticas e de hidrofobicidade. A interação de CPPs com membranas leva à sua translocação através desta, enquanto que os AMPs as costumam danificar. Uma nova classificação engloba CPPs e AMPs num grande grupo denominado por péptidos com atividade membranar ( do Inglês “Membrane active peptides” - MAPs). Neste trabalho foram desenhados três péptidos, o GA10 (GAGAGAGAGAGAGAGAGAGA), o LK10 (LKLKLKLKLKLKLKLKLKLK) e o LE10 (LELE LELELELELELELELE), para o estudo das suas interações com membranas lipídicas, por simulações de dinâmica molecular (do Inglês “Molecular Dynamics” - MD) e ensaios laboratoriais, nomeadamente ensaios de caracterização físico-química e de fluorescência com lipossomas, ensaios de citotoxicidade e testes de atividade antimicrobiana. O objetivo de estudo é contribuir para uma melhor caracterização dos mecanismos responsáveis pela atuação de MAPs em membranas celulares. Os resultados de MD mostraram uma interação evidente do péptido LE10 com as membranas simuladas, que leva à dobra das membranas à volta deste péptido. Essa interação foi confirmada pelos estudos laboratoriais. O péptido LE10, carregado negativamente, apresenta características de CPP (translocação através de membranas) e de AMP (disrupção membranar) nos estudos realizados, apontando para que a carga positiva possa não ser uma propriedade fundamental, e para que a classificação de MAPs seja a mais adequada a estes tipos de péptidos. Tendo em conta as propriedades aqui demonstradas pelo péptido LE10, modificações na sua estrutura podem torná-lo num vector promissor na entrega dirigida de fármacos.The interaction between peptides and membranes plays a fundamental role in many processes, such as in membrane proteins functions, in antimicrobial activity, in viral infections, and in process that leads to diseases like the neurodegenerative Alzheimer and Parkinson. Cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are generally defined as small cationic peptides that have the property of interacting with lipidic membranes, in a process driven by their electrostatic and hydrophobicity features. The interaction with CPPs is known to lead to its translocation across the membrane, while with AMPs by lead to membrane damage. One new classification includes the CPPs and AMPs in one big group denominated by membrane active peptides (MAPs). This work reports the design of three peptides, the GA10 (GAGAGAGAGAGAGAGAGAGA), the LK10 (LKLKLKLKLKLKLKLKLKLK) and the LE10 (LELELELELELELELELELE) for the study of their interactions with lipidic membranes, by molecular dynamics (MD) simulations and laboratory assays, namely assays of physicochemical characterization and of fluorescence of liposomes, cytotoxic assays and antimicrobial activity tests. The objective of this work is to contribute towards a better characterization of the mechanisms behind the MAPs actuation over cell membranes. The MD results showed one evident interaction of the LE10 peptide with the simulated membranes, which leads to the membrane bending around the peptide. This interaction was also confirmed by laboratory experiments. The LE10 peptide, negatively charged, shows features of CPP (translocation through membranes) and AMP (membrane disruption) in the performed studies, indicating that the positive charge may not be a fundamental property, with the classification of MAP being more suitable for this type of peptides. Based on the LE10 properties here demonstrated, modifications in its structure may make it a very promising tool as a drug delivery vector

Molecular dynamics simulations together with experimental studies reveal strong membrane activity of a small peptide

251st American Chemical Society National Meeting & Exposition - Computers in ChemistryCell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs) are generally defined as small cationic peptides with the ability to interact with lipidic membranes, in a process driven by electrostatic and hydrophobic processes. The interaction with CPPs is known to lead to its translocation across the membrane, while with AMPs lead to membrane damage. Here we present one synthetic anionic peptide, which strongly interacts with model membranes, showing properties of the two peptide classes, namely the translocation through lipidic membranes on a mechanism usually described for cationic CPPs and membrane destabilization like AMPs promote. These properties were shown through molecular dynamic studies, experimental studies with liposomes and mammalian cells in vitro. Based on the peptide properties here demonstrated, small modifications in its structure could make it a very promising tool for drug delivery.info:eu-repo/semantics/publishedVersio

Hair keratin molecular dynamics models

251st American Chemical Society National Meeting & Exposition - Computers in ChemistryThe keratin is a key element of the hair, nails and skin in vertebrates. Understand the keratin features such as its assembling in the mentioned structures, its interaction with some compounds or mechanical properties is of great interest in the fight against some diseases or in the development and optimization of cosmetic products. Molecular dynamics modelling is the only technique able to provide information at atomic and molecular level in a dynamic way, which can greatly help in the study of these features. However there are only a few studies using molecular dynamics simulations. This is likely to the non-existence of full length crystallographic structure models of keratin. In the few works published about keratin using molecular dynamics simulations the authors had to design and build the computational keratin model, to make the simulations of interest. This work addresses some keratin models developed, from its physicochemical properties to the correlation of the simulations results with experimental data. Our recently developed computational model of a truncated hair protofibril (8 chains of keratin), which was able to predict the increase of peptide absorption by hair shaft in response to alcohol based formulations, is also discussed.info:eu-repo/semantics/publishedVersio

Hair keratin molecular dynamics studies

EJIBCE 2016 - IV Encontro de Jovens Investigadores de Biologia Computacional EstruturalThe keratin is a key element of the hair, nails and skin in vertebrates. Understand the keratin features such as its assembling in the mentioned structures, its interaction with some compounds or mechanical properties is of great interest in the fight against some diseases or in the development and optimization of cosmetic products. Although molecular dynamics simulations provides unique information at molecular level there are only a few studies using this technique on the study of keratin. This is likely the result of the non-existence of full length keratin crystallographic model. In the few works published about keratin using molecular dynamics simulations the authors had to design and build the computational keratin model, to make the simulations of interest. This work addresses some molecular dynamics studies about hair keratin, from the physicochemical properties of the molecular models to the correlation of the simulations results with experimental data. Our work on this field, with recently developed computational models of hair fibers, is also discussed. We built molecular dynamics models able to reproduce in simulations some phenomena observed in experimental assays, providing important information at molecular level about the mechanisms that lead to the experimental results.info:eu-repo/semantics/publishedVersio

Hair keratin molecular dynamics studies

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] The keratin is a key element of the hair, nails and skin in vertebrates. Understand the keratin features such its assembling in the mentioned structures, its interactions with some compounds or mechanical properties is of great interest in the fight against some diseases or in the development and optimization of cosmetic products. Although molecular dynamics (MD) simulations provides unique information at molecular level in a dynamic way, there are only a few studies using this technique on the study of keratin. This is likely the result of the nonexistence of full length keratin crystallographic model. In the few published works the authors had to design and build the computational keratin model to perform the simulations of interest. [...]info:eu-repo/semantics/publishedVersio

Straighten curly hair with keratin peptides

Book of Abstracts of CEB Annual Meeting 2017[Excerpt] Chemical straightening curly human hair fibres involves the use of strong reducing agents at alkaline pHs. These treatments damage the hair fibre, reduce the cross‐linking density and decrease hair’s  physico mechanical properties [1]. Human hair is made of keratin and the fixation of fibres shape involve the reduction and reformation of new disulphide bonds between keratin molecules. t is known that cysteine has been applied as a reducing agent for the substitution of environmentally harmful chemicals[2]. [...]info:eu-repo/semantics/publishedVersio

Keratin molecular dynamics models

The keratin is a key element of the hair, nails and skin in vertebrates. Understand the keratin features such as its assembling in the mentioned structures, its interaction with some compounds or mechanical properties is of great interest in the fight against some diseases or in the development and optimization of cosmetic products. Molecular dynamics modelling is the only technique able to provide information at atomic and molecular level in a dynamic way, which can greatly help in the study of these features. However there are only a few studies using molecular dynamics simulations. This is likely to the non-existence of full length crystallographic structure models of keratin. In the few works published about keratin using molecular dynamics simulations the authors had to design and build the computational keratin model, to make the simulations of interest. This work addresses the different keratin models developed, from its physicochemical properties to the correlation of the simulations results with experimental data. One big computational model of a truncated protofibril (8 chains of keratin), which was able to predict the increase of peptide absorption by hair shaft in response to alcohol based formulations, is also discussed

Fusion proteins with chromogenic and keratin binding modules

The present research relates to a fusion protein comprising a chromogenic blue ultramarine protein (UM) bound to a keratin-based peptide (KP). The KP-UM fusion protein explores UM chromogenic nature together with KP affinity towards hair. For the first time a fusion protein with a chromogenic nature is explored as a hair coloring agent. The KP-UM protein colored overbleached hair, being the color dependent on the formulation polarity. The protein was able to bind to the hair cuticle and even to penetrate throughout the hair fibre. Molecular dynamics studies demonstrated that the interaction between the KP-UM protein and the hair was mediated by the KP sequence. All the formulations recovered the mechanical properties of overbleached hair and KP-UM proved to be safe when tested in human keratinocytes. Although based on a chromogenic non-fluorescent protein, the KP-UM protein presented a photoswitch phenomenon, changing from chromogenic to fluorescent depending on the wavelength selected for excitation. KP-UM protein shows the potential to be incorporated in new eco-friendly cosmetic formulations for hair coloration, decreasing the use of traditional dyes and reducing its environmental impact.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469/2019 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684) and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. We also acknowledge the strategic program UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through Fundação para a Ciência e a Tecnologia (FCT) and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalização (POCI). Ana Tinoco, Filipa Gonçalves and Carla Silva thanks FCT for funding the scholarships with the references SFRH/BD/114035/2015, FRH/BD/114684/2016 and IF/00186/2015, respectively.info:eu-repo/semantics/publishedVersio

Internalization of methotrexate conjugates by folate receptor-α

The folate antagonist methotrexate is a cytotoxic drug used in the treatment of several cancer types. Methotrexate entry into the cell is mediated by two main transport systems: the reduced folate carrier and membrane-associated folate receptors. These transporters differ considerably in their mechanism of (anti)folate uptake, substrate specificity and tissue specificity. Although the mechanism of action of the reduced folate carrier is fairly well-established, that of the folate receptor has remained doubtful. The development of specific folate receptor-targeted antifolates would be accelerated if additional mechanistic data becomes available. In this work, we used two fluorescent-labeled conjugates of methotrexate, differently linked at the terminal groups, to clarify the uptake mechanism by the folate receptor-α. The results demonstrate the importance of methotrexate amino groups in the interaction with the folate receptor-α.E.A. (SFRH/BD/122952/2016) and J.N. (SFRH/BD/121673/ 2016) hold scholarships from the Portuguese Foundation for Science and Technology (FCT). This study was supported by the FCT under the scope of the strategic funding of the UID/ BIO/04469/2013 unit and the COMPETE 2020 (POCI-010145-FEDER-006684) and BioTecNorte operation (NORTE01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020Programa Operacional Regional do Norte. This work has also received funding from the European Union 7th Framework Programme (FP7/2007−2013), under Grant Agreement NMP4-LA-2009228827 NANOFOL, and the Horizon 2020 research and innovation program under Grant Agreement NMP-06-2015683356 FOLSMART.info:eu-repo/semantics/publishedVersio

Stratum corneum lipid matrix with unusual packing: a molecular dynamics study

The skin is an effective barrier against the external elements being the stratum corneum, with its lipid matrix surrounding the corneocytes, considered the major player responsible for its low permeability. The use of computational models to study the transdermal delivery of compounds have a huge potential to improve this research area, but requires reliable models of the skin components. In this work, we developed molecular dynamics models with a coarse-grained resolution, of the stratum corneum lipid matrix and the sebum. We developed the lipid matrix model with unusual lipid packing configuration as some recent works support. The simulation results show that this configuration is stable and may help to explain the low permeability of stratum corneum. The sebum simulations showed that this oily skin product can also play a significant role in the transdermal delivery of drugs.We thank the Portuguese Foundation for Science and Technology (FCT) for providing the grant for part of Egipto Antunes Ph.D. studies (scholarship SFRH/BD/122952/2016), and to also support this work under the scope of the strategic funding of UID/BIO/04469/2019 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norte. We want to thank the access of Minho University “SeARCH” ("Services and Advanced Research Computing with HTC/HPC clusters") cluster. We also thank Tarsila Castro for its technical advice and text revision.info:eu-repo/semantics/publishedVersio