Structural Basis for Agonistic Activity and Selectivity toward Melatonin Receptors hMT1 and hMT2

: Glaucoma, a major ocular neuropathy originating from a progressive degeneration of retinal ganglion cells, is often associated with increased intraocular pressure (IOP). Daily IOP fluctuations are physiologically influenced by the antioxidant and signaling activities of melatonin. This endogenous modulator has limited employment in treating altered IOP disorders due to its low stability and bioavailability. The search for low-toxic compounds as potential melatonin agonists with higher stability and bioavailability than melatonin itself could start only from knowing the molecular basis of melatonergic activity. Thus, using a computational approach, we studied the melatonin binding toward its natural macromolecular targets, namely melatonin receptors 1 (MT1) and 2 (MT2), both involved in IOP signaling regulation. Besides, agomelatine, a melatonin-derivative agonist and, at the same time, an atypical antidepressant, was also included in the study due to its powerful IOP-lowering effects. For both ligands, we evaluated both stability and ligand positioning inside the orthosteric site of MTs, mapping the main molecular interactions responsible for receptor activation. Affinity values in terms of free binding energy (ΔGbind) were calculated for the selected poses of the chosen compounds after stabilization through a dynamic molecular docking protocol. The results were compared with experimental in vivo effects, showing a higher potency and more durable effect for agomelatine with respect to melatonin, which could be ascribed both to its higher affinity for hMT2 and to its additional activity as an antagonist for the serotonin receptor 5-HT2c, in agreement with the in silico results

Towards a high-throughput microfluidic single-cell proteomic platform for analysing patient blood samples

A critical driver in the development of single-cell analysis platforms has been the recognition that cellular heterogeneity is crucial to understanding disease. Single cell proteomics offer significant insights of cellular function; however, currently suffers from low-throughput. The work outlined here presents the development and application of several single-cell protein analysis systems. Each aim to address technological gaps regarding throughput, cell selectivity and amenability to processing samples directly from patients. To achieve higher-throughput, we have developed the CellWell platform, a high-density microwell array which can capture thousands of cells within minutes; however, posed challenges relating to the simultaneous lysis of these cells. We developed a facile method to produce surface microelectrodes to achieve single-cell lysis on-chip, but the demanding surface chemistry requirements imposed by the necessity to support simultaneously both the microelectrodes and single-molecule antibody microarrays proved difficult to overcome. Instead, we investigated how implementing semi-permeable hydrogel-based microwells could overcome these issues. To assay cells in patient blood samples with the CellWell, pre-processing is necessary. With a clinical setting in mind, it would be advantageous to process raw samples directly from patients with little or no off-chip pre-processing. To address this, we develop our methodology into the Hydrodynamic Trapping Centrifugal Release (HTCR) chip which is specifically designed to isolate cancer cells from patient liquid biopsies. The HTCR implements a method by which cells can be easily released from hydrodynamic traps and subsequently moved to isolated compartments. We conclude in validating the single-molecule single-cell method using fluorescence and immunofluorescence microscopy. While necessary to validate our single-molecule approach, we also show that the equivalence of these measurements of the steady-state distribution of protein abundance can be exploited to pave the way for absolute quantitation by fluorescence and immunofluorescence microscopy.Open Acces

Computational Modeling of Protein Kinases: Molecular Basis for Inhibition and Catalysis

Protein kinases catalyze protein phosphorylation reactions, i.e. the transfer of the γ-phosphoryl group of ATP to tyrosine, serine and threonine residues of protein substrates. This phosphorylation plays an important role in regulating various cellular processes. Deregulation of many kinases is directly linked to cancer development and the protein kinase family is one of the most important targets in current cancer therapy regimens. This relevance to disease has stimulated intensive efforts in the biomedical research community to understand their catalytic mechanisms, discern their cellular functions, and discover inhibitors. With the advantage of being able to simultaneously define structural as well as dynamic properties for complex systems, computational studies at the atomic level has been recognized as a powerful complement to experimental studies. In this work, we employed a suite of computational and molecular simulation methods to (1) explore the catalytic mechanism of a particular protein kinase, namely, epidermal growth factor receptor (EGFR); (2) study the interaction between EGFR and one of its inhibitors, namely erlotinib (Tarceva); (3) discern the effects of molecular alterations (somatic mutations) of EGFR to differential downstream signaling response; and (4) model the interactions of a novel class of kinase inhibitors with a common ruthenium based organometallic scaffold with different protein kinases. Our simulations established some important molecular rules in operation in the contexts of inhibitor-binding, substrate-recognition, catalytic landscapes, and signaling in the EGFR tyrosine kinase. Our results also shed insights on the mechanisms of inhibition and phosphorylation commonly employed by many kinases

Structural Dynamics and Allosteric Signaling in Ionotropic Glutamate Receptors

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate excitatory neurotransmission events in the central nervous system. All distinct classes of iGluRs (AMPA, NMDA, Kainate) are composed of an N-terminal domain (NTD) and a ligand-binding domain (LBD) in their extracellular domain, a transmembrane domain (TMD) and an intracellular carboxy-terminal domain (CTD). Ligand binding to the LBD facilitates ion channel activation. The NTDs modulate channel gating allosterically in NMDA receptors (NMDARs). A similar function of the NTD in AMPA receptors (AMPARs) is still a matter of debate. Taking advantage of recently resolved structures of the NTD and the intact AMPAR, the main focus of this dissertation is a comprehensive examination of iGluR NTD structural dynamics, ligand binding and allosteric potential of AMPARs. We use a multiscale, multi-dimensional approach using coarse-grained network models and all-atom simulations for structural analyses and information theoretic approaches for examination of evolutionary correlations. Our major contribution has been the characterization of the global motions favored by iGluR NTD architecture. These intrinsic motions favor ligand binding in NMDAR NTDs and are also shared by other iGluR NTDs. We also identified structural determinants of flexibility in AMPARs and confirmed their role through in silico mutants. The overall similarity in collective dynamics among iGluRs hints at a putative allosteric capacity of non-NMDARs and has propelled the elucidation of interdomain and intersubunit coupling in the intact AMPAR. To this end, we identified “effector” and “sensor” regions in AMPARs using a perturbation-response technique. We identified potentially functional residues that enable information propagation between effector regions and proposed an efficient mechanism of allosteric communication based on a combination of tools including network models, graph theoretical methods and sequence analyses. Finally, we assessed the “druggability” of iGluR NTDs using molecular dynamics simulations in the presence of probe molecules containing fragments shared by drug-like molecules. Based on our study, we offer key insights into the ligand-binding landscape of iGluR NTD monomers and dimers, and we also identify a novel ligand-binding site in AMPAR dimers. These findings open an avenue of searching for molecules able to bind to iGluR NTDs and allosterically modulate receptor activity

Targeting human aquaporin function : physiological and chemical approaches

Tese de doutoramento, Farmácia (Bioquímica), Universidade de Lisboa, Faculdade de Farmácia, 2018Aquaporins (AQPs) are a group of small membrane proteins belonging to a highly conserved family of membrane proteins called MIPs (Major Intrinsic Proteins) that are responsible for the bidirectional transport of wate (orthodox aquaporins) and also small uncharged solutes (aquaglyceroporins) across cell membranes, in response to osmotic or solute gradients. Rapid water flux across membranes is crucial to maintain the water homeostasis in many epithelia and endothelia involved in fluid transport. In addition, due to the unique ability of aquaglyceroporins to transport glycerol in addition to water, they have important roles in glycerol metabolism and skin hydration in non-fluid transporting tissues such as skin, fat and liver. The thesis introduction (Chapter 1) presents an overview of aquaporins structure, their main biological functions and related pathologies, with special emphasis on the so far described mechanisms of regulation. In the first part of this thesis (Chapter 2), we report the discovery of a new role for Aquaporin-5 (AQP5, an orthodox aquaporin) in adipocyte biology, where Aquaporin-7 (AQP7, an aquaglyceroporin) has been the mainly characterized protein in adipose tissue responsible for glycerol efflux. A better understanding of aquaporin regulation and gating would allow manipulation of their activity facilitating the identification of new putative modulators. A cellular model optimized to assess the function of aquaporins and discriminate individually each isoform, instead of mammalian cells where more than one isoform is usually expressed, is a useful tool to study aquaporin regulation. The second part of this thesis (Chapter 3) is dedicated to the functional characterization of different mammalian aquaporin isoforms (AQP3, AQP5, AQP7 and AQP10), using a yeast heterologous expression system devoided of endogenous aquaporins, a background where analysis is unlikely to be compromised by the co-expression of other aquaporin isoforms. Using the stopped-flow technique to evaluate the channel permeability for water and for glycerol, we were able to disclose gating mechanisms of aquaporin isoforms, being given special emphasis to the regulation by pH and phosphorylation. In the third part of this thesis (Chapter 4), a screening of several small gold compounds as inhibitors for Aquaporin-3 (AQP3, an aquaglyceroporin) was performed aiming at identifying new modulators with potential therapeutic use.A água possui um papel crucial para a vida devido às suas propriedades únicas. Todos os processos bioquímicos e fisiológicos de um organismo dependem da presença de água, sendo esta o componente fundamental na manutenção da homeostase celular. Nas células eucarióticas, a água encontra-se distribuída pelos vários compartimentos intracelulares separados entre si por membranas intracelulares e do meio extracelular pela membrana plasmática. Estas membranas de composição bilipídica são normalmente impermeáveis à maioria dos solutos polares e/ou carregados, cuja passagem é facilitada através de canais membranares específicos. No entanto estas membranas são bastante permeáveis à água, sendo então propostas três vias de transporte: por difusão simples, por transporte passivo associado ao transporte de iões e solutos e por canais específicos para a água. Atualmente sabe-se que a maioria das células de um organismo possui proteínas específicas – as aquaporinas – que conferem à membrana uma permeabilidade à água de cerca de 5 a 10 vezes superior às membranas que não possuem estas proteínas. Devido às suas características estruturais, as aquaporinas permitem um rápido transporte bidirecional de água, seletivo e regulado, em resposta a gradientes osmóticos, ao mesmo tempo que previnem a passagem de protões e iões através da membrana plasmática. Em mamíferos, são conhecidas até à data treze isoformas (AQP0-AQP12) que são classificadas em três grupos de acordo com a sua sequência primária, localização celular e seletividade em 1) aquaporinas ortodoxas, primariamente seletivas à água; 2) aquagliceroporinas, para além de água também transportam pequenos solutos neutros, como glicerol e ureia; e 3) super-aquaporinas, que são encontradas em membranas intracelulares e possuem menor homologia. No entanto, a lista de substâncias que são capazes de permear as diferentes aquaporinas tem aumentado ao longo do tempo. Recentemente, para além de água e glicerol, foi também descrito o transporte facilitado através de algumas isoformas de arsenito, amoníaco e peróxido de hidrogénio. Devido à grande diversidade de tecidos onde são encontradas as aquaporinas, o seu papel de facilitar o transporte de água e/ou solutos através das membranas plasmáticas é importante em vários processos fisiológicos, tais como: secreção de fluido glandular, mecanismo de concentração urinária, excitabilidade neuronal, metabolismo dos lípidos, hidratação epidérmica e balanço de água no cérebro. A observação do fenótipo de ratinhos geneticamente modificados com knock-out de determinadas aquaporinas revelou funções fisiológicas muito importantes no aparecimento e desenvolvimento de várias patologias, como epilepsia, edema cerebral, glaucoma, cancro e obesidade. No Capítulo 1 é apresentada uma introdução geral que visa proporcionar um conhecimento abrangente sobre as principais funções das aquaporinas humanas e patologias associadas, dando especial atenção aos diferentes mecanismos de regulação já conhecidos. Na primeira parte dos resultados desta tese (Capítulo 2), através da construção de linhas celulares de pré adipócitos de ratinho 3T3-L1 com diferentes níveis de expressão da Aquaporin-5 (cenário de ganho e perda de função) foi possível estabelecer um novo e determinante papel desta aquaporina na diferenciação dos adipócitos. Na segunda parte dos resultados desta tese (Capítulo 3), pretendeu-se usar um sistema de expressão heteróloga em Saccharomyces cerevisiae (S. cerevisiae) para permitir avaliar de forma individual a função de cada aquaporina. A levedura S. cerevisiae é considerada um valioso sistema de expressão heteróloga para estudar inúmeras proteínas devido à elevada homologia funcional entre esta e os eucariontes superiores, incluindo mamíferos. Pelo facto de existir uma grande biblioteca de estirpes disponíveis, ser de fácil manipulação genética, ser pouco dispendioso em comparação com as culturas de células animais e poderem ser testadas uma variedade de condições externas, este sistema oferece condições experimentais ótimas para estudar a especificidade e regulação das aquaporinas. Após a expressão e confirmação da sua localização celular, procedeu-se à caracterização da função de cada isoforma, utilizando a técnica de interrupção brusca de fluxo, seguindo a variação de volume celular por fluorescência quando se introduz uma perturbação no meio extracelular. Os fluxos de água através da membrana celular causados por gradientes de pressão osmótica (de solutos impermeantes ou permeantes) provocam alterações transitórias de volume, até se atingir um novo volume final de equilíbrio osmótico. A velocidade com que as alterações de volume ocorrem e o tempo que a célula leva a re-estabelecer o seu novo equilíbrio osmótico dependem diretamente das características intrínsecas de transporte da membrana, em particular da quantidade de canais específicos para a água e para o soluto em questão. No Capítulo 3 foram estudadas quatro isoformas diferentes (AQP3, AQP5, AQP7 e AQP10) e os seus mecanismos de regulação por pH e fosforilação foram revelados pela primeira vez. Vários esforços têm vindo a ser feitos com o intuito de desenvolver possíveis fármacos para tratamento das aquaporinopatias, mas até agora nenhum composto se revelou qualificado para estudos in vivo, quer pela sua fraca solubilidade quer pela sua baixa capacidade de inibição. Na terceira parte dos resultados desta tese (Capítulo 4), deu-se especial atenção à descoberta de novos compostos organometálicos, inibidores da função da Aquaporina-3, que poderão ser usados para benefício clínico na prevenção ou tratamento das várias patologias associadas.Fundação para a Ciência e a Tecnologia (FCT), SFRH/BD/52384/2013, Programa de Doutoramento Medical Biochemistry and Biophysics Doctoral Programme (M2B-PhD

Myxococcus xanthus: an approach to phenotyping complex biofilms

The genotype-to-phenotype (G2P) problem is a fundamental challenge in understanding how genetic information controls the collective phenotypic outputs of multicellular organisms. To address this problem, this thesis focuses on Myxococcus xanthus, a model organism widely used for studying social behavior and morphological differentiation in bacteria. The social behaviors of M. xanthus are mediated by complex gene regulatory networks, making it an excellent model organism for studying the G2P problem. This thesis presents two studies that provide valuable insights into the G2P problem in M. xanthus. The first study investigates the dynamics of biofilm morphogenesis, an essential process for bacteria survival, using image capture and analysis techniques from custom-designed microscopes. Biofilms are complex structures composed of different cell types and gene expression patterns. The study produced a topological map of the process of biofilm formation in wild-type M. xanthus, which allowed the identification and characterization of even subtle mutations associated with different mutations with statistical significance. Stochastic variation was mapped, and this methodology allowed us to distinguish previously non-distinguishable genotypes of M. xanthus using phenotype data. The approach used in this study has the potential to enhance our understanding of the genetic and environmental factors that contribute to the development of M. xanthus and other organisms. The second study employs a common garden approach to investigate the impact of transcriptional regulators on development in M. xanthus. By recording subtle differences in traditional phenotype assays across a library of mutant transcriptional regulators, the study shows there are uncharacterized regulatory genes that play significant roles in regulating biofilm dynamics and gene expression during development. The study identified sigma factors that have an impact on sporulation fitness and characterized the effects of these regulators on the development pathways. The findings of this study supply a deeper understanding of the G2P problem in M. xanthus and could have broader implications for understanding the development of other organisms. By investigating the complex interplay between genotype and phenotype, this thesis aims to shed light on fundamental mechanisms underlying multicellular development, and the potential for these findings to be applied in the fields of biotechnology and medicine

Light-Enabled Identification of the Neuronal Substrates of Alkylphenol Anesthetics

General anesthetics are a critical class of drugs in modern medicine; however, the precise mechanisms by which they cause unconsciousness and unwanted side effects are largely undefined. In order to understand pharmacologic mechanisms of anesthetic action, drug interactions with macromolecular substrates and the subsequent functional consequences must be characterized. Analogs of general anesthetics that function as photolabels have been developed to assist in the identification of molecular targets. One such photolabel, meta-azi-propofol (AziPm), is an analog of the clinically used alkylphenol anesthetic propofol. In this work, AziPm is employed in a variety of experiments that aim to identify molecular substrates of propofol. Two proteins identified as propofol targets are more thoroughly examined: (1) the sirtuin deacetylase SIRT2 and (2) the mitochondrial voltage-dependent anion channel (VDAC). The binding sites of propofol on these proteins, and the in vitro functional consequences of propofol binding, are determined. Also described are the molecular interactions of VDAC with a separate ligand, cholesterol, which shares a binding site with propofol. In addition to molecular studies, a novel in vivo photolabeling technique, called optoanesthesia, that utilizes AziPm is introduced, and the behavioral phenotype induced by optoanesthesia in Xenopus laevis tadpoles is characterized. Finally, optoanesthesia is demonstrated with other ligands, including a photoactive analog of an anthracene anesthetic, and mechanistic insight into the pharmacology of this anthracene is revealed