Chromatographic design for the purification of recombinant human membrane COMT

Several studies suggest that membrane form of catechol-O-methyltransferase OMT (MB-COMT) is the main responsible for O-methylation at physiologically low concentrations of catecholamines. Despite this, until now no studies have been allowed the total isolation of MB-COMT. Then, a sustainable chromatographic step should be developed in order to obtain significant quantities of active and pure enzyme for posterior application on biochemical, kinetic and structural studies. For the first time, we intend to compare the performance of three hydrophobic adsorbents (Butyl-, Epoxy- and Octyl-sepharose) in the purification of human membrane-bound COMT (hMBCOMT) from crude Brevibacillus choshinensis cell lysates. The hydrophobic matrices were evaluated in terms of selectivity, binding and elution conditions. Results show that the isolation of MB-COMT was possible using 375 mM monosodium phosphate concentrations for its adsorption. The hMB-COMT, as the soluble form, was found to elute at four different fractions at 0.25; 0.7 and 1% Triton X-100. Preliminary chromatographic trials indicate that hMBCOMT may be isolated on octyl- with mild salt conditions but on epoxy- were required high salt concentrations to complete enzyme adsorption. Thereby and in conclusion, in this work and for the first time was possible the total isolation of significant amounts of MB-COMT on a single chromatography step with high selectivity. Although successful applications of Hydrophobic Interaction Chromatography (HIC) in the purification of membrane proteins are uncommon, in this work we prove that traditional hydrophobic matrices can open a promising unexplored field in order to fulfill specific requirements for kinetic and pharmacological trials

Human vitreous proteome in vitreoretinal diseases

Vitreous, also termed vitreous body or vitreous humor, is a transparent fluid that fills the posterior cavity of the eye, surrounded by the neurosensorial retina, and lens. For a long time, the vitreous was not appreciated for its role in health and disease, and its function was thought to be merely structural. Nevertheless, the analysis of vitreous proteome has gained a growing interest in recent years. These studies proved that vitreous is highly complex and biologically more active than initially thought. As a matter of fact, changes in vitreous proteome reflect the physiological and pathological state of the eye, and, therefore, it is the ideal matrix for studying vitreoretinal diseases. Although the search for sensitive and specific vitreous biomarkers in ocular disease has not been successful so far, the analysis of vitreous proteome has been seen to be promising in elucidating some of the pathological mechanisms underlying vitreoretinal diseases. In this project, several gel-based and gel-free techniques were developed and applied for the analysis of vitreous proteome in retinal detachment (RD), diabetic retinopathy (DR), and age-related macular degeneration (AMD). Since the early proteomic studies, two-dimensional gel electrophoresis (2DE) has been the preferential method for the separation and identification of vitreous proteins. If combined with more sensitive detection techniques, refined gel image processing, and proper sample preparation, 2DE is still a valuable tool for high-resolution separation and routine analysis of proteoforms. Despite technological advances, 2DE of biological fluids, such as vitreous, remains a major challenge. Therefore, in the first part of this work, an artificial neural network was applied to optimize the recovery of vitreous proteins and their detection by 2DE analysis through the combination of several solubilizing agents (CHAPS, Genapol, DTT, IPG buffer) and physical parameters (temperature and total voltage). Using a mathematical model created by ANN, both the protein recovery and the number of spots detected in 2DE gels were significantly improved. The optimized response (580 spots) represents a 2.4-fold improvement over the standard conditions applied for vitreous analysis by 2DE. Our results clearly indicate that it is crucial to combine appropriate amounts of solubilizing agents to improve the extraction, solubilization, and detection of vitreous proteins, and to obtain well-resolved gels. Beyond that, our results also indicate that physical parameters have a significant influence on isoelectric focusing and, thereby, should be adjusted and monitored. When working with biological fluids, it is also important to reduce their complexity before 2DE analysis to facilitate the detection of low-abundant proteins, and to increase the detected in the gel increased 1.3-fold over the optimal output refined by the ANN model, with an average of 761 spots detected in vitreous from different vitreoretinopathies, including rhegmatogenous retinal detachment (RRD) and the proliferative diabetic retinopathy (PDR). In the second task of this Ph.D. project, the performance of gel-free proteomic techniques combined with stable-isotope labeling was tested for the analysis of vitreous samples in RRD. RRD is a potentially blinding condition characterized by a physical separation between the neurosensory retina and retinal pigment epithelium. Vitreous has a central role in the onset of RRD, which may be triggered by vitreous liquefaction. It reduces the vitreoretinal adhesion, leading to the accumulation of vitreous fluid in subretinal space, and, subsequently, to the physical separation between the neuronal retina and the retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur in the eye, providing additional information about the molecular mechanisms underlying RRD pathogenesis. In this study, the proteome of vitreous collected from patients with RRD was analyzed and compared to epimacular membranes (MEM) using iTRAQ reagents (Isobaric tags for relative and absolute quantitation) combined with analysis by two-dimensional liquid chromatography coupled to tandem mass spectrometry (2D-LC-MS/MS). Using this strategy, we identified 6078 peptides corresponding to 1030 proteins, with 2613 out of these corresponded to unique peptides. Overall, 150 proteins were found differentially expressed in the RRD vitreous, including 96 overexpressed and 54 underexpressed. Among overexpressed proteins, several glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1), glucose transporters (GLUT-1), and protease inhibitors (metalloproteinase inhibitor 1, plasminogen activator inhibitor 1) are regulated by hypoxia-inducible factor-1 (HIF-1), which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the overexpression of proteins of carbon metabolism or molecular chaperones or, among others, suggests that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses, e.g. the activation of the HIF-1 signaling pathway. In the third task, a label-free quantitative (LFQ) method was applied to analyze the vitreous proteome in PDR and dry AMD. DR and AMD are leading causes of visual impairment and blindness in people aged 50 years or older in middle-income and industrialized countries. Although Anti-VEGF therapies have improved the management of neovascular AMD (nAMD) and PDR, no treatment options exist for dry AMD. Therefore, quantitative proteomics can help to recognize the biological mechanisms underlying these pathologies and to find new potential biomarkers and/or pharmaceutical targets. For this purpose, the proteome of vitreous collected from patients with PDR (n=4) were compared to dry AMD (n=4) and epiretinal membranes (ERM) (n=4) using an LFQ method that combines a fractionation by short SDS–polyacrylamide gel electrophoresis and analysis by LC-MS/MS. A total of 680 proteins were identified, of which 586 were identified using the software search engine MASCOT and 580 using MaxQuant. Subsequently, post hoc tests, hierarchical clustering, and multiple t-tests were performed for differentiating the three disease groups in terms of protein expression based on their intensity. Post hoc tests revealed that 96 proteins are capable of differentiating among the different groups, whereas 118 proteins (17 up- and 101 down-regulated) were found differentially regulated in PDR compared to ERM and 95 proteins (10 up- and 85 down-regulated) in PDR compared to dry AMD. Functional enrichment analysis indicates that these underexpressed proteins are correlated to pathways/ biological processes, such as extracellular matrix (ECM) disassembly and organization, platelet degranulation, lysosomal degradation, cell adhesion, and central nervous system development. In turn, mediators of complement and coagulation cascades and acute-phase inflammatory responses were found enriched in PDR vitreous, reinforcing the role of these pathways in its pathogenesis of PDR. For last, some potential biomarkers were selected according to iTRAQ and LFQ experiments and validated by multiple reaction monitoring (MRM) in a larger set of vitreous samples. Therefore, we develop a scheduled MRM method for the analysis of 35 proteins in vitreous samples collected from patients with ERM (n=21), DR/PDR (n=20), AMD (n=11), and RRD (with and without proliferative vitreoretinopathy) (n=13). Of these, 26 proteins have been shown the potential to differentiate between different disease groups according to MRM results and respective receiver operating characteristic curves. Complement and coagulation components (C6, C8B, prothrombin), acute-phase proteins (alpha-1-antichymotrypsin), adhesion molecules (galectin-3-binding protein), ECM components (opticin), and neurodegeneration biomarkers (beta-amyloid, amyloid-like protein 2) stand out as the more efficient biomarkers to discriminate among the different disease groups. In conclusion, several gel-based and gel-free strategies were develop ed and implemented for the preparation and analysis of the proteome of vitreous in different vitreoretinal diseases. Concerning the gel-based method, a mathematical model created by ANN provided an effective 2DE protocol for high-resolution analysis of vitreous proteome, which can be advantageous for analysis of specific proteoforms, including different isoforms and post-translational modified proteins. On the other hand, high-throughput methods, such as iTRAQ and LFQ, provided a more in-depth analysis of vitreous proteome. In these techniques, we identified 1030 proteins by iTRAQ and 680 by LFQ, some of them have not been previously identified. Even more relevant is the fact that vitreous analysis using these techniques provided new insights on the pathogenesis of RRD, PDR, and AMD. Beyond that, they provided fundamental information regarding potential biomarkers, which enabled the successful validation of 26 proteins by MRM. Nevertheless, it must be taken into consideration that vitreous biomarkers cannot be used for regular diagnosis due to invasive sampling. However, they can be candidates for new pharmaceutical targets and, when the samples are obtained as part of the clinical routine, be used for the prognosis of the patient's disease evolution and/or to predict the proper response to treatment.O vítreo, também denominado por corpo vítreo ou humor vítreo, é um fluido transparente que preenche a cavidade posterior do olho entre a retina neurossensorial e o cristalino. Durante muitos anos, o papel do vítreo na saúde e na doença foi negligenciado, pensando-se que a sua função era meramente estrutural. No entanto, tem-se registado um crescente interesse pela análise do proteoma vítreo nos últimos anos. Estes estudos comprovaram que o vítreo é altamente complexo e biologicamente mais ativo do que se pensava inicialmente. De facto, alterações a nível do proteoma do vítreo refletem o estado fisiológico e patológico do olho e, portanto, esta é a matriz ideal para o estudo das doenças vitreorretinianas. Embora a procura de biomarcadores no vítreo, mais sensíveis e específicos para cada patologia ocular, não tenha sido bem-sucedida até o momento, a análise do proteoma vítreo mostrou-se promissora na elucidação de alguns dos mecanismos patológicos subjacentes a estas patologias. Neste projeto, diversas técnicas proteómicas baseadas na separação de proteínas em gel de poliacrilamida (gel-based proteomics) ou no fracionamento de péptidos por cromatografia líquida (gel-free proteomics) foram desenvolvidas e aplicadas para a análise do proteoma do vítreo no descolamento da retina (RD), na retinopatia diabética (DR) e na degeneração macular relacionada com a idade (AMD). Desde os primeiros estudos em proteómica, a eletroforese bidimensional do gel (2DE) foi o método preferencial para a separação e a identificação de proteínas do vítreo. A 2DE é uma ferramenta valiosa para a separação com elevada resolução e análise de rotina de proteoformas, principalmente se for combinada com técnicas de deteção mais sensíveis, com um processamento de imagem mais refinado e com uma preparação adequada das amostras. Portanto, na primeira parte deste trabalho, aplicou-se uma rede neural artificial (ANN) para a otimização da extração de proteínas do vítreo e da sua análise por 2DE, através da combinação de vários agentes solubilizantes (CHAPS, Genapol, DTT, tampão IPG) e parâmetros físicos (temperatura e voltagem total). Pela aplicação de um modelo matemático criado por ANN, a extração de proteínas e o número de spots detetados após a sua análise por 2DE melhoram significativamente. A resposta otimizada (580 spots detetados) representa um incremento melhoria de 2,4 vezes comparando com as condições padrão utilizadas no desenho experimental inicial. Os resultados alcançados indicam claramente que é crucial combinar as concentrações adequadas de agentes solubilizantes para melhorar a extração, solubilização e a deteção das proteínas do vítreo, assim como para obter géis bem resolvidos. Para além disso, os nossos resultados também indicam que os parâmetros físicos têm uma influência significativa na focagem isoelétrica e, por esta razão, devem ser ajustados e monitorizados neste tipo de análise. Quando se trabalha com fluidos biológicos também é importante reduzir a sua complexidade antes da análise por 2DE, de modo a facilitar a deteção de proteínas pouco abundantes e a aumentar a cobertura do proteoma. Após a remoção da albumina e da imunoglobulina G, o número de proteínas detetadas no gel aumentou 1,3 vezes quando comparado com o ponto ótimo do modelo proposto por ANN, com uma média de 761 spots detetados no vítreo em doenças vitreorretinianas, como, por exemplo, o descolamento regmatogénico da retina (RRD) ou a retinopatia diabética proliferativa (PDR). Na segunda tarefa deste projeto de doutoramento, testou-se a performance das técnicas de marcação isobárica, na análise de amostras de vítreo de RRD. O RRD é uma das causas de cegueira e é caracterizado por uma separação física entre a retina neurossensorial e o epitélio pigmentar da retina (RPE). O vítreo tem um papel central no aparecimento do RRD, que pode ser provocado pela liquefação do vítreo. Esta reduz a adesão vitreorretiniana, conduzindo assim à acumulação de líquido do vítreo no espaço subretinal, e, consequentemente, à separação física entre a retina e o RPE. Assim, a proteómica quantitativa pode contribuir para a compreensão das alterações que ocorrem no olho, providenciando uma informação complementar sobre os mecanismos moleculares subjacentes à patogénese do RRD. No presente estudo, o proteoma do vítreo recolhido de doentes com RRD foi analisado e comparado com amostras de vítreo de membranas epimaculares (MEM) usando reagentes iTRAQ (Isobaric tags for relative and absolute quantitation) em combinação com análise por Cromatografia Líquida Bidimensional acoplada à Espectrometria de Massa em Tandem (2D-LC-MS/MS). A análise destas amostras por LC-MS/MS resultou na identificação de 6078 péptidos relativos a 1030 proteínas, 2613 dos quais correspondem a péptidos únicos. Das proteínas identificadas, um total de 150 estava diferencialmente expressa no vítreo de doentes com RRD, incluindo 96 proteínas sobreexpressas e 54 subexpressas. Entre as sobreexpressas encontraram-se várias enzimas glicolíticas (frutose-bifosfato aldolase A, gama-enolase e fosfoglicerato cinase 1), transportadores de glicose (GLUT-1), e inibidores de proteases (inibidor da metaloproteinase 1, inibidor do ativador de plasminogénio 1) que são regulados pelo fator induzido por hipóxia (HIF-1), o que sugere que a via de sinalização HIF-1 pode ser activada em resposta à RRD. Além disso, a acumulação no vítreo de proteínas intracelulares dos fotorreceptores, incluindo fosducina, rodopsina e S-arrestina, ou da vimentina revela que a RRD leva a uma degeneração significativa das células fotorreceptoras. No entanto, a sobreexpressão de proteínas envolvidas no metabolismo do carbono ou chaperones moleculares, entre outras, indica que diversos mecanismos são ativados em resposta ao RRD de forma a promover a sobrevivência das células retinianas através de respostas celulares complexas, como por exemplo, a ativação da via de sinalização HIF-1. Na terceira tarefa, aplicou-se um método quantitativo label-free (LFQ) para analisar o proteoma do vítreo na PDR e na forma seca da AMD (dry AMD). DR e AMD são as principais causas de deficiência visual e cegueira em indivíduos com idade igual ou superior a 50 anos, em países industrializados ou de rendimento médio. Embora as terapias direcionadas à inibição do fator de crescimento vascular (VEGF) tenham melhorado o tratamento da forma neovascular da AMD (nAMD) e da PDR, neste momento não existem opções terapêuticas para a AMD seca. Portanto, a proteómica quantitativa pode contribuir para o conhecimento dos mecanismos biológicos subjacentes a estas patologias e a encontrar novos potenciais biomarcadores e/ou alvos terapêuticos. Com esta finalidade, o proteoma do vítreo recolhido de doentes com PDR (n=4) foi comparado com o de doentes com AMD seca (n=4) e com membranas epiretinianas (ERM) (n=4) utilizando um método LFQ, que combina o fracionamento “curto” por eletroforese desnaturante em gel de poliacrilamida e análise por LC-MS/MS. Foram identificadas 680 proteínas, das quais 586 foram identificadas com recurso ao software MASCOT e 580 com o software MaxQuant. Posteriormente, foram realizados testes post hoc, métodos hierárquicos para análise de agrupamento de dados e testes t múltiplos para diferenciar os três grupos de doenças em termos de expressão proteica com base na sua intensidade. Os testes post hoc revelaram que 96 proteínas são capazes de diferenciar entre os diferentes grupos, enquanto 118 proteínas (17 para sobreexpressas e 101 subexpressas) foram identificados como diferencialmente expressas na PDR em comparação com doentes com ERM e 95 proteínas (10 sobreexpressas e 85 subexpressas) em comparação com os doentes com AMD seca. A análise de enriquecimento funcional indica que estas proteínas subexpressas estão correlacionadas com vias/processos biológicos, como reorganização da matriz extracelular (ECM), desgranulação das plaquetas, digestão intracelular nos lisossomas, adesão celular e desenvolvimento do sistema nervoso central. Por sua vez, os resultados indicam que o vítreo de doentes com PDR é enriquecido em mediadores dos sistemas de complemento e coagulação e da fase aguda da inflamação, reforçando o papel destas vias na sua patogénese. Por último, alguns potenciais biomarcadores foram selecionados de acordo com os resultados obtidos na quantificação do proteoma do vítreo por iTRAQ e LFQ e validados pela monitorização de múltiplas reações (MRM) num maior número de amostras de vítreo. Assim, desenvolveu-se um método de MRM scheduled para a análise de 35 proteínas, em amostras de vítreo recolhidas de doentes com ERM (n=21), DR/PDR (n=20), AMD (n=11) e RRD (com e sem vitreorretinopatia proliferativa) (n=13). Desta forma, 26 proteínas demostraram potencial para discriminar entre os diferentes grupos de doenças de acordo com os resultados obtidos no MRM e as respectivas curvas ROC (receiver operating characteristic curve). Componentes das cascatas do complemento e coagulação (C6, C8B, protrombina), proteínas de fase aguda (alfa-1-antiquimotripsina), moléculas de adesão (proteína galectina-3), componentes da ECM (opticina) e biomarcadores de neurodegeneração (beta-amilóide, proteína tipo-precursora amilóide 2) destacam-se como os biomarcadores mais eficientes para discriminar entre os diferentes grupos de doenças. Em conclusão, foram desenvolvidas e implementadas diversas estratégias para a preparação e análise do proteoma do vítreo em diferentes doenças vitreorretinianas, baseadas na separação por 2DE ou por LC. Em relação ao método baseado na separação em gel, um modelo matemático criado por ANN permitiu o desenvolvimento de um protocolo altamente eficiente para a análise de elevada resolução do proteoma do vítreo por 2DE, o que pode ser vantajoso para a detecção de proteoformas específicas, incluindo diferentes isoformas e proteínas com modificações pós-traducionais. Por outro lado, métodos de alta produtividade, como iTRAQ e LFQ, proporcionaram uma análise mais aprofundada do proteoma do vítreo. Nestas técnicas, foram identificadas 1030 proteínas pela técnica de iTRAQ e 680 por LFQ, sendo que algumas não tinham sido identificadas anteriormente. Ainda mais relevante é o fato de que a análise do proteoma do vítreo, com base nestas técnicas, forneceu novos perspetivas sobre a patogénese do RRD, PDR e AMD. Além disso, estes estudos forneceram informações fundamentais sobre potenciais biomarcadores, o que permitiu a validação de 26 proteínas por MRM. No entanto, deve ter-se em consideração que os biomarcadores encontrados no vítreo não podem ser utilizados para um diagnóstico médico regular, devido ao modo de recolha invasivo deste tipo de amostras. Porém, estes podem ser candidatos a novos alvos farmacêuticos e, quando as amostras são obtidas como parte da rotina clínica, podem ser usados para o prognóstico da evolução da doença e/ou para prever a resposta adequada ao tratamento

SARS-CoV-2 accessory proteins involvement in inflammatory and profibrotic processes through IL11 signaling

SARS-CoV-2, the cause of the COVID-19 pandemic, possesses eleven accessory proteins encoded in its genome. Their roles during infection are still not completely understood. In this study, transcriptomics analysis revealed that both WNT5A and IL11 were significantly up-regulated in A549 cells expressing individual accessory proteins ORF6, ORF8, ORF9b or ORF9c from SARS-CoV-2 (Wuhan-Hu-1 isolate). IL11 is a member of the IL6 family of cytokines. IL11 signaling-related genes were also differentially expressed. Bioinformatics analysis disclosed that both WNT5A and IL11 were involved in pulmonary fibrosis idiopathic disease and functional assays confirmed their association with profibrotic cell responses. Subsequently, data comparison with lung cell lines infected with SARS-CoV-2 or lung biopsies from patients with COVID-19, evidenced altered profibrotic gene expression that matched those obtained in this study. Our results show ORF6, ORF8, ORF9b and ORF9c involvement in inflammatory and profibrotic responses. Thus, these accessory proteins could be targeted by new therapies against COVID-19 disease

Vitreous Humor Proteome: Targeting Oxidative Stress, Inflammation, and Neurodegeneration in Vitreoretinal Diseases

Oxidative stress is defined as an unbalance between pro-oxidants and antioxidants, as evidenced by an increase in reactive oxygen and reactive nitrogen species production over time. It is important in the pathophysiology of retinal disorders such as diabetic retinopathy, age-related macular degeneration, retinal detachment, and proliferative vitreoretinopathy, which are the focus of this article. Although the human organism’s defense mechanisms correct autoxidation caused by endogenous or exogenous factors, this may be insufficient, causing an imbalance in favor of excessive ROS production or a weakening of the endogenous antioxidant system, resulting in molecular and cellular damage. Furthermore, modern lifestyles and environmental factors contribute to increased chemical exposure and stress induction, resulting in oxidative stress. In this review, we discuss the current information about oxidative stress and the vitreous proteome with a special focus on vitreoretinal diseases. Additionally, we explore therapies using antioxidants in an attempt to rescue the body from oxidation, restore balance, and maximize healthy body function, as well as new investigational therapies that have shown significant therapeutic potential in preclinical studies and clinical trial outcomes, along with their goals and strategic approaches to combat oxidative stress

Assessment of Aptamer as a Potential Drug Targeted Delivery for Retinal Angiogenesis Inhibition

AT11-L0 is an aptamer derivative of AS1411 composed of G-rich sequences that can adopt a G-quadruplex (G4) structure and target nucleolin (NCL), a protein that acts as a co-receptor for several growth factors. Hence, this study aimed to characterize the AT11-L0 G4 structure and its interaction with several ligands for NCL targeting and to evaluate their capacity to inhibit angiogenesis using an in vitro model. The AT11-L0 aptamer was then used to functionalize drug-associated liposomes to increase the bioavailability of the aptamer-based drug in the formulation. Biophysical studies, such as nuclear magnetic resonance, circular dichroism, and fluorescence titrations, were performed to characterize the liposomes functionalized with the AT11-L0 aptamer. Finally, these liposome formulations with the encapsulated drugs were tested on the human umbilical vein endothelial cell (HUVEC) model to assess their antiangiogenic capacity. The results showed that the AT11-L0 aptamer–ligand complexes are highly stable, presenting melting temperatures from 45 °C to 60 °C, allowing for efficient targeting of NCL with a KD in the order of nM. The aptamer-functionalized liposomes loaded with ligands C8 and dexamethasone did not show cytotoxic effects in HUVEC cells compared with the free ligands and AT11-L0, as assessed by cell viability assays. AT11-L0 aptamer-functionalized liposomes encapsulating C8 and dexamethasone did not present a significant reduction in the angiogenic process when compared with the free ligands. In addition, AT11-L0 did not show anti-angiogenic effects at the concentrations tested. However, C8 shows potential as an angiogenesis inhibitor, which should be further developed and optimized in future experiments