Diffusion and association processes in biological systems: theory, computation and experiment

Macromolecular diffusion plays a fundamental role in biological processes. Here, we give an overview of recent methodological advances and some of the challenges for understanding how molecular diffusional properties influence biological function that were highlighted at a recent workshop, BDBDB2, the second Biological Diffusion and Brownian Dynamics Brainstorm

Protein folding, protein structure and the origin of life: Theoretical methods and solutions of dynamical problems

Theoretical methods and solutions of the dynamics of protein folding, protein aggregation, protein structure, and the origin of life are discussed. The elements of a dynamic model representing the initial stages of protein folding are presented. The calculation and experimental determination of the model parameters are discussed. The use of computer simulation for modeling protein folding is considered

Molecular Mechanism of Early Amyloid Self-Assembly Revealed by Computational Modeling

Protein misfolding followed by the formation of aggregates, is an early step in the cascade of conformational changes in a protein that underlie the development of several neurodegenerative diseases, including Alzheimer’s and Parkinson’s diseases. Efforts aimed at understanding this process have produced little clarity and the mechanism remains elusive. Here, we demonstrate that the hairpin fold, a structure found in the early folding intermediates of amyloid b, induces morphological and stability changes in the aggregates of Aβ(14-23) peptide. We structurally characterized the interactions of monomer and hairpin using extended molecular dynamics (MD) simulations, which revealed a novel intercalated type complex. These finding suggest that folding patterns of amyloid proteins define the aggregation pathway. Computational analysis was then used to characterize the dimerization of full-length Aβ peptide and reveal their dynamic properties. Aβ dimers did not show β-sheet structures, as one may expect based on the known structures of Aβ fibrils, rather dimers are stabilized by hydrophobic interactions in the central hydrophobic regions. Comparison between Aβ40 and Aβ42 showed that overall, the dimers of both alloforms exhibit similar interaction strengths. However, the interaction patterns are significantly different. A novel aggregation pathway, able to describe aggregation at physiologically relevant concentrations, was elucidated when aggregation of amyloid proteins was performed in presence of surfaces. Computational analysis revealed that interaction of a monomer with the surface is accompanied by the structural transition of the monomer; which can then facilitate binding of another monomer and form a dimer. Compared to our previous data we observed an almost five-fold faster dimer formation. Further investigation of the surface-mediated aggregation revealed that lipid membranes promote aggregation of a-syn protein. MD simulations demonstrate that a-syn monomers change conformation upon interaction with the bilayers. On POPS, a-syn monomer protrudes from the surface. This conformation on POPS dramatically facilitates assembly of a dimer that remains stable over the entire simulation period. These findings are in line with experimental data. Overall, the studies described in this thesis provide the structural basis for the early stages of the misfolding and aggregation process of amyloid proteins

Systems Biology of Protein Secretion in Human Cells: Multi-omics Analysis and Modeling of the Protein Secretion Process in Human Cells and its Application.

Since the emergence of modern biotechnology, the production of recombinant pharmaceutical proteins has been an expanding field with high demand from industry. Pharmaceutical proteins have constituted the majority of top-selling drugs in the pharma industry during recent years. Many of these proteins require post-translational modifications and are therefore produced using mammalian cells such as Chinese Hamster Ovary cells. Despite frequent improvements in developing efficient cell factories for producing recombinant proteins, the natural complexity of the protein secretion process still poses serious challenges for the production of some proteins at the desired quantity and accepted quality. These challenges have been intensified by the growing demands of the pharma industry to produce novel products with greater structural complexity,\ua0\ua0as well as increasing expectations from regulatory authorities in the form of new quality control criteria to guarantee product safety.This thesis focuses on different aspects of the protein secretion process, including its engineering for cell factory development and analysis in diseases associated with its deregulation. A major part of this thesis involved the use of HEK293 cells as a human model cell-line for investigating the protein secretion process by generating different types of omics data and developing a computational model of the human protein secretion pathway. We compared the transcriptomic profile of cell lines producing erythropoietin (EPO; as a model secretory protein) at different rates to identify key genes that potentially contributed to higher rates of protein secretion. Moreover, by performing a transcriptomic comparison of cells producing green fluorescent protein (GFP; as a model non-secretory protein) with EPO producers, we captured differences that specifically relate to secretory protein production. We sought to further investigate the factors contributing to increased recombinant protein production by analyzing additional omic layers such as proteomics and metabolomics in cells that exhibited different rates of EPO production. Moreover, we developed a toolbox (HumanSec) to extend the reference human genome-scale metabolic model (Human1) to encompass protein-specific reactions for each secretory protein detected in our proteomics dataset. By generating cell-line specific protein secretion models and constraining the models using metabolomics data, we could predict the top host cell proteins (HCPs) that compete with EPO for metabolic and energetic resources.\ua0Finally,\ua0based on the detected patterns of changes in our multi-omics investigations combined with a protein secretion sensitivity analysis using the metabolic model, we identified a list of genes and pathways that potentially play a key role in recombinant protein production and could serve as promising candidates for targeted cell factory design.In another part of the thesis, we studied the link between the expression profiles of genes involved in the protein secretory pathway (PSP) and various hallmarks of cancer. By\ua0implementing a dual approach involving differential expression analysis and eight different machine learning algorithms, we investigated the expression changes in secretory pathway components across different cancer types to identify PSP genes whose expression was associated with tumor characteristics. We demonstrated that a combined machine learning and differential expression approach have a complementary nature and could highlight key PSP components relevant to features of tumor pathophysiology that may constitute potential therapeutic targets

Disorder dynamic in cell: protein unfolding, molecular crowding and their combined action in intracellular aqueous two-phase systems formation

Dissertação de mestrado em Bioquímica Aplicada, ramo BiotecnologiaInside the cell, the heterogeneous distribution of macromolecules can produce regions of different density, also known as membraneless organelles. These organelles are formed by phase separation, creating a specific microenvironment for the occurrence of localized biochemistry. The mechanism by which this phase separation occurs is still controversial, although various experimental evidences suggest a major role of intermolecular interactions between intrinsically disordered proteins (IDPs), proteins that display high structural flexibility crucial for this dynamic association. On the other hand, there is an increased evidence that globular proteins present high structural flexibility, where not only the native states but also the partially and globally unfolded states can have biological function. Accordingly, it is proposed that the intermolecular interactions between partial/globally unfolded states of globular proteins and other proteins could also lead to phase separation. The analysis of such hypothesis involved the in vitro mimicking of heat stress condition, a physiological situation that encompasses both the intracellular phase separation and thermal unfolding. To accomplish this, myoglobin was selected as a model to study the thermal unfolding and an in vitro model for phase separation was used, based on PEG/Dextran aqueous two-phase systems (ATPS). In addition, and to closer mimic the protein´s native environment, namely, the intracellular ions, KCl was added to the model. For comparative purposes, the effect of additional four different salts in the myoglobin stability (thermal and chemical) was evaluated. The analysis of the results for each salt suggests a thermal destabilization induced by Debye-Hückel screening, whereas in the case of chemical denaturation, the effects of salts followed their position in the Hofmeister series. Structural data from circular dichroism (CD) and fluorescence resonance energy transfer (FRET) analysis indicate a perturbation of the myoglobin structure both in the native and unfolded state, with the latter being distinct for thermal or chemical unfolding. The results regarding the proposal pointed out for an earlier phase separation of PEG/dextran systems above certain concentrations of myoglobin. The main reason for this effect emerges from the direct interactions between the myoglobin unfolded states and PEG, indicating that unfolded states of globular proteins can alter the behavior of phase separation, namely, by decreasing the concentration needed for its occurrence. Moreover, an additional contribution of the solvent was observed, suggesting that the interplay between the proteins and their surrounding water must be considered when studying the mechanism of intracellular phase separation. Collectively, the present results strengthen the proposed hypothesis, opening new biochemical perspectives for the occurrence of such phenomenon inside the cells.No interior da célula, a distribuição heterogénea de macromoléculas pode levar à formação de regiões com densidade diferente, designadas por organelos sem membrana. A formação destes organelos tem por base a separação de fases, criando microambientes específicos para a ocorrência de diversas reações bioquímicas. O mecanismo pelo qual esta separação de fases ocorre é controverso, apesar de várias evidências experimentais apontarem para uma relevância acrescida das interações intermoleculares entre proteínas intrinsecamente desordenadas. Estas proteínas possuem uma elevada flexibilidade estrutural, crucial para a sua associação dinâmica e consequente separação de fases. Por outro lado, estudos recentes indicam que as proteínas globulares apresentam elevada flexibilidade estrutural, onde, além dos estados nativos, também os estados parcial/totalmente desenroladas exibem função biológica. Deste modo, é proposto que interações intermoleculares entre estados parcialmente ou totalmente desenrolados de proteínas globulares com outras proteínas possam originar separação de fases. A analise desta hipótese baseou-se na mimetização in vitro de uma situação de stress térmico, uma vez que este ultimo envolve simultaneamente a ocorrência de separação de fases e o desenrolamento de proteínas. Para tal, a mioglobina foi selecionada como proteína modelo para estudar a desnaturação térmica e um modelo de separação de fases in vitro foi aplicado, baseado em sistemas de duas-fases aquosas PEG/Dextrano. Adicionalmente, e de modo a mimetizar mais fielmente o ambiente nativo das proteínas (nomeadamente, os sais intracelulares), KCl foi adicionado ao modelo. Para análise comparativa, o efeito de outros quatro sais na estabilidade térmica e química foi explorado. Os resultados do efeito individual de cada sal sugerem uma destabilização térmica da mioglobina devido a efeitos de Debye-Hückel screening enquanto, na desnaturação química, os sais influenciam a estabilidade da proteína de acordo com a posição dos mesmos na serie Hofmeister. Os resultados do efeito do desenrolamento das globulares proteínas na separação de fases indicam que esta ocorre para concentrações mais baixas de polímeros, com o aumento da concentração de mioglobina. Tal observação pode ser explicada com base nas interações adicionais entre o PEG e os estados desenrolados da mioglobina, apoiando deste modo, o envolvimento de estados desenrolados de proteínas globulares na separação de fases. Além disso, foi também observado um possível efeito adicional do solvente, sugerindo que as interações entre proteínas e as moléculas de agua nas proximidades devem ser consideradas quando se estuda o mecanismo molecular que leva a formação de separação de fases dentro da célula.The present work was performed in the Department of Chemistry, University of Minho, Braga, Portugal and founded by the Foundation for Science and Technology, FCT-Portugal, through direct financial support of Centre of Chemistry of University of Minho (CQUM) (projects UID/QUI/00686/2013 and UID/QUI/00686/2016)