A bidomain threshold model of propagating calcium waves

We present a bidomain fire-diffuse-fire model that facilitates mathematical analysis of propagating waves of elevated intracellular calcium (Ca) in living cells. Modelling Ca release as a threshold process allows the explicit construction of travelling wave solutions to probe the dependence of Ca wave speed on physiologically important parameters such as the threshold for Ca release from the endoplasmic reticulum (ER) to the cytosol, the rate of Ca resequestration from the cytosol to the ER, and the total [Ca] (cytosolic plus ER). Interestingly, linear stability analysis of the bidomain fire-diffuse-fire model predicts the onset of dynamic wave instabilities leading to the emergence of Ca waves that propagate in a back-and-forth manner. Numerical simulations are used to confirm the presence of these so-called "tango waves" and the dependence of Ca wave speed on the total [Ca]. The original publication is available at www.springerlink.com (Journal of Mathematical Biology)

Release currents of IP₃ receptor channel clusters and concentration profiles

We simulate currents and concentration profiles generated by Ca2+ release from the endoplasmic reticulum (ER) to the cytosol through IP3 receptor channel clusters. Clusters are described as conducting pores in the lumenal membrane with a diameter from 6 nm to 36 nm. The endoplasmic reticulum is modeled as a disc with a radius of 1–12 mm and an inner height of 28 nm. We adapt the dependence of the currents on the trans Ca2+ concentration (intralumenal) measured in lipid bilayer experiments to the cellular geometry. Simulated currents are compared with signal mass measurements in Xenopus oocytes. We find that release currents depend linearly on the concentration of free Ca2+ in the lumen. The release current is approximately proportional to the square root of the number of open channels in a cluster. Cytosolic concentrations at the location of the cluster range from 25 μM to 170 μM. Concentration increase due to puffs in a distance of a few micrometers from the puff site is found to be in the nanomolar range. Release currents decay biexponentially with timescales of < 1 s and a few seconds. Concentration profiles decay with timescales of 0.125–0.250 s upon termination of release.

Improving protein yield from mammalian cells by manipulation of stress response pathways

Monoclonal antibodies are a class of therapeutic that is an expanding area of the lucrative biopharmaceutical industry. These complex proteins are predominantly produced from large cultures of mammalian cells; the industry standard cell line being Chinese Hamster Ovary (CHO) cells. A number of optimisation strategies have led to antibody titres from CHO cells increasing by a hundred-fold, and it has been proposed that a further bottleneck in biosynthesis is in protein folding and assembly within the secretory pathway. To alleviate this bottleneck, a CHO-derived host cell line was generated by researchers at the pharmaceutical company UCB that stably overexpressed two critical genes: XBP1, a transcription factor capable of expanding the endoplasmic reticulum and upregulating protein chaperones; and Ero1α, an oxidase that replenishes the machinery of disulphide bond formation. This host cell line, named CHO-S XE, was confirmed to have a high yield of secreted antibody. The work presented in this thesis further characterises CHO-S XE, with the aim of using the information gained to lead the generation of novel host cell lines with more optimal characteristics than CHO-S XE. In addition to antibodies, it was found that CHO-S XE had improved production of two other secreted proteins: one with a simple tertiary structure and one complex multi-domain protein; and higher levels of a number of endogenous protein chaperones. As a more controlled system of gene expression to unravel the specific roles of XBP1 and Ero1α in the secretory properties of CHO-S XE, CHO cells with inducible overexpression of XBP1, Ero1α, or a third gene involved in the Unfolded Protein Response, GADD34, were generated. From these cell lines, it was shown that more antibody was secreted by cells with induced overexpression of XBP1; however, Ero1α and GADD34 overexpression did not improve antibody yield. Further investigation revealed that endogenous XBP1 splicing was downregulated in the presence of an abundance of the active form of XBP1. This result indicated a novel aspect of the regulation of the activity of IRE1, the stress-induced endoribonuclease responsible for XBP1 splicing. Overall, the work described in this thesis confirms that the overexpression of XBP1 has an enhancing effect on the secretory properties of CHO cells; information which could contribute to the development of host cells with a greater capacity for antibody production.

An investigation into the folding and assembly of engineered antibodies and novel antibody formats

Monoclonal antibodies and novel antibody formats are currently one of the principal therapeutic in the biopharmaceutical industry worldwide and are widely used in the treatment of autoimmune diseases and cancer. It is for this reason that the productivity and quality of antibody production requires improvement; specifically investigations into the engineering of antibodies and any issues that may arise from the production of these therapeutics. The work presented in this thesis describes an investigation into the folding and assembly of seven antibodies plus the novel antibody format FabFv. IgG is comprised of two identical HCs and two identical LCs. The folding process of immunoglobulin is controlled by the CH1 domain within the HC. The CH1 domain remains in a disordered state and is sequestered by BiP in the endoplasmic reticulum. Upon the addition of a folded CL domain, BiP is displaced, the CH1 domain is able to fold and the complete IgG protein can then be secreted from the cell. The results presented in this thesis however, have outlined an additional mechanism for the folding of the CH1 domain. We have shown that the CH1 domain is able to fold in the absence of LC resulting in the secretion of HC dimers in a VH dependent manner. The proposed mechanism for the secretion of HC dimers suggests that some VH domains can interact with each other in order to bring the CH1 domains in close proximity to enable folding to occur. As HC dimer secretion is a hindrance in antibody production, this result has highlighted an engineering target to improve antibody yield. Examination of the folding of IgG4 with the variable region A33 has revealed the inability to secrete LC dimers, cleavage of the HC during expression and secretion of HC dimers in the Fab, FabFv and full length forms. The attributes described have also been shown to be variable region dependent. This has introduced a new concept that the variable domain is important in determining the expression and secretion of antibodies and their individual chains. Pulse chase and 2D gel electrophoresis analysis of the novel antibody format FabFv has revealed that the folding and expression of the LC and HC causes multimeric species of FabFv to be secreted, as opposed to the monomeric form which is the desired therapeutic. Our hypothesis is that this process occurs via a LC dependent mechanism. The proposed hypothesis suggests that further engineering to the LC could diminish the formation and secretion of FabFv multimers. The results from these investigations can be applied to increase the productivity of therapeutics and increase the biological understanding of the domain interactions of IgG during folding, assembly and secretion.

Exploring the Function and Regulation of Arabidopsis EIN2 in Ethylene Signaling

Ethylene is an essential plant hormone involved in nearly all stages of plant growth and development. EIN2 (ETHYLENE INSENSITIVE2) is a master positive regulator in the ethylene signaling pathway, consisting of an N-terminal domain and a C-terminal domain. The EIN2 N-terminal domain localizes to the endoplasmic reticulum (ER) membrane and shows sequence similarity to Nramp metal ion transporters. The cytosolic C-terminal domain is unique to plants and signals downstream. There have been several major gaps in our knowledge of EIN2 function. It was unknown how the ethylene signal gets relayed from the known upstream component CTR1 (CONSTITUTIVE RESPONSE1) a Ser/Thr kinase at the ER, to EIN2. How the ethylene signal was transduced from EIN2 to the next downstream component transcription factor EIN3 (ETHYLENE INSENSITIVE3) in the nucleus was also unknown. The N-terminal domain of EIN2 shows homology to Nramp metal ion transporters and whether EIN2 can also function as a metal transporter has been a question plaguing the ethylene field for almost two decades. Here, EIN2 was found to interact with the CTR1 protein kinase, leading to the discovery that CTR1 phosphorylates the C-terminal domain of EIN2 in Arabidopsis thaliana. Using tags at the termini of EIN2, it was deduced that in the presence of ethylene, the EIN2 C-terminal domain is cleaved and translocates into the nucleus, where it could somehow activate downstream ethylene responses. The EIN2 C-terminal domain interacts with nuclear proteins, RTE3 and EER5, which are components of the TREX-2 mRNA export complex, although the role of these interactions remains unclear. The EIN2 N-terminal domain was found to be capable of divalent metal transport when expressed in E. coli and S. cerevisiae leading to the hypothesis that metal transport plays a role in ethylene signaling. This hypothesis was tested using a novel missense allele, ein2 G36E, substituting a highly conserved residue that is required for metal transport in Nramp proteins. This G36E substitution did not disrupt metal ion transport of EIN2, but the ethylene insensitive phenotype of this mutant indicates that the EIN2 N-terminal domain is important for positively regulating the C-terminal domain. The defect of the ein2 G36E mutant does not prevent proper expression or subcellular localization, but might affect protein modifications. The ein2 G36E allele is partially dominant, mostly likely displaying haploinsufficiency. Overexpression of the EIN2 N-terminal domain in the ein2 G36E mutant did not rescue ethylene insensitivity, suggesting the N-terminal domain functions in cis to regulate the C-terminal domain. These findings advance our knowledge of EIN2, which is critical to understanding ethylene signaling.

Poli-líquidos iônicos e complexos solúveis de Eu3+ e Tb3+ : bioimageamento celular seletivo

Dissertação (mestrado)—Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2015.

Interacção de complexos de vanádio-citrato com a bomba de cálcio de retículo sarcoplasmático

Dissertação de mest. em Biotecnologia, Departamento de Química e Bioquímica da Faculdade de Ciêicias e Tecnologia, Univ. do Algarve, 2004

New insights into plant salt acclimation: the roles of vesicle trafficking and reactive oxygen species signalling in mitochondria and the endomembrane system

In this study, we investigated the cellular and molecular mechanisms that regulate salt acclimation. The main objective was to obtain new insights into the molecular mechanisms that control salt acclimation. Therefore, we carried out a multidisciplinary study using proteomic, transcriptomic, subcellular and physiological techniques. We obtained a Nicotiana tabacum BY-2 cell line acclimated to be grown at 258 mM NaCl as a model for this study. The proteomic and transcriptomic data indicate that the molecular response to stress (chaperones, defence proteins, etc.) is highly induced in these salt-acclimated cells. The subcellular results show that salt induces sodium compartmentalization in the cell vacuoles and seems to be mediated by vesicle trafficking in tobacco salt-acclimated cells. Our results demonstrate that abscisic acid (ABA) and proline metabolism are crucial in the cellular signalling of salt acclimation, probably regulating reactive oxygen species (ROS) production in the mitochondria. ROS may act as a retrograde signal, regulating the cell response. The network of endoplasmic reticulum and Golgi apparatus is highly altered in salt-acclimated cells. The molecular and subcellular analysis suggests that the unfolded protein response is induced in salt-acclimated cells. Finally, we propose that this mechanism may mediate cell death in salt-acclimated cells.

Structural and ultrastructural characteristics of interrenal gland and chromaffin cell of matrinxa, Brycon cephalus Gunther 1869 (Teleostei-Characidae)

This work presents the structure and ultrastructure of the interrenal gland and chromaffin cells, as well as the morphology of the head kidney of Brycon cephalus, the head kidney is composed of fused bilateral lobes located anterior to the swim bladder and ventrolateral to the spinal column, the parenchyma revealed lympho-haematopoietic tissue, melano-macrophage centres, interrenal gland and chromaffin cells. The interrenal gland consisted of cords or strands of cells grouped around the posterior cardinal vein and their branches. Chromaffin cells are found in small groups, closely associated with the interrenal gland and/or under the endothelium of the posterior cardinal vein. So far, the ultrastructural analysis has revealed only one interrenal cell type which contained abundant smooth endoplasmic reticulum and numerous mitochondria with tubulo-vesicular cristae, characteristic of steroid-producing cells. Two types of chromaffin cells were observed. The first type was characterized by the presence of vesicles with round, strongly electron-dense granules, which were eccentrically located, Such cells were interpreted as noradrenaline cells, Meanwhile, cells which contained smaller vesicles and electron-lucent granules, with a small halo separating the granule from the vesicular limiting membrane, were identified as adrenaline cells.

Understanding Co-translational Protein Targeting and Lithium Dendrite Formation through Free Energy Simulations and Coarse-Grained Models

We describe the application of alchemical free energy methods and coarse-grained models to study two key problems: (i) co-translational protein targeting and insertion to direct membrane proteins to the endoplasmic reticulum for proper localization and folding, (ii) lithium dendrite formation during recharging of lithium metal batteries. We show that conformational changes in the signal recognition particle, a central component of the protein targeting machinery, confer additional specificity during the the recognition of signal sequences. We then develop a three-dimensional coarse-grained model to study the long-timescale dynamics of membrane protein integration at the translocon and a framework for the calculation of binding free energies between the ribosome and translocon. Finally, we develop a coarse-grained model to capture the dynamics of lithium deposition and dissolution at the electrode interface with time-dependent voltages to show that pulse plating and reverse pulse plating methods can mitigate dendrite growth.

Host cell remodelling in malaria parasites: a new pool of potential drug targets

When in their human hosts, malaria parasites spend most of their time housed within vacuoles inside erythrocytes and hepatocytes. The parasites extensively modify their host cells to obtain nutrients, prevent host cell breakdown and avoid the immune system. To perform these modifications, malaria parasites export hundreds of effector proteins into their host cells and this process is best understood in the most lethal species to infect humans, Plasmodium falciparum. The effector proteins are synthesized within the parasite and following a proteolytic cleavage event in the endoplasmic reticulum and sorting of mature proteins into the correct vesicular trafficking pathway, they are transported to the parasite surface and released into the vacuole. The effector proteins are then unfolded before extrusion across the vacuole membrane by a unique translocon complex called Plasmodium translocon of exported proteins. After gaining access to the erythrocyte cytoplasm many effector proteins continue their journey to the erythrocyte surface by utilising various membranous structures established by the parasite. This complex trafficking pathway and a large number of the effector proteins are unique to Plasmodium parasites. This pathway could, therefore, be developed as new drug targets given that protein export and the functional role of these proteins are essential for parasite survival. This review explores known and potential drug targetable steps in the protein export pathway and strategies for discovering novel drug targets.

Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein

Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer's disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.

Élucidation et identification des différents interacteurs impliqués dans le mécanisme de régulation du LDLR par la protéine PCSK9

Résumé : Les maladies cardiovasculaires représentent la principale cause de mortalité mondiale, soit le tiers des décès annuels selon l’Organisation mondiale de la Santé. L’hypercholestérolémie, caractérisée par une élévation des niveaux plasmatiques de lipoprotéines de faible densité (LDL), est l’un des facteurs de risque majeur pour les maladies cardiovasculaires. La proprotéine convertase subtilisine/kexine type 9 (PCSK9) joue un rôle essentiel dans l’homéostasie du cholestérol sanguin par la régulation des niveaux protéiques du récepteur LDL (LDLR). PCSK9 est capable de se lier au LDLR et favorise l’internalisation et la dégradation du récepteur dans les lysosomes. L’inhibition de PCSK9 s’avère une cible thérapeutique validée pour le traitement de l’hypercholestérolémie et la prévention des maladies cardiovasculaires. Par contre, plusieurs mécanismes responsables de la régulation et la dégradation du complexe PCSK9-LDLR n’ont pas encore été complètement caractérisés comme la régulation par la protéine annexin A2 (AnxA2), un inhibiteur endogène de PCSK9. De plus, plusieurs évidences suggèrent la présence d’une ou plusieurs protéines, encore inconnues, impliquées dans le mécanisme d’action de PCSK9. Celles-ci pourraient réguler l’internalisation et le transport du complexe PCSK9-LDLR vers les lysosomes. Les objectifs de cette thèse sont de mieux définir le rôle et l’impact de l’AnxA2 sur la protéine PCSK9 en plus d’identifier de nouveaux partenaires d’interactions de PCSK9 pour mieux caractériser son mécanisme d’action sur la régulation des niveaux de LDLR. Nous avons démontré que l’inhibition de PCSK9 par l’AnxA2 extracellulaire s’effectue via sa liaison aux domaines M1+M2 de la région C-terminale de PCSK9 et nous avons mis en évidence les premières preuves d’un contrôle intracellulaire de l’AnxA2 sur la traduction de l’ARNm de PCSK9. Nos résultats révèlent une liaison de l’AnxA2 à l’ARN messager de PCSK9 qui cause une répression traductionnelle. Nous avons également identifié la protéine glypican-3 (GPC3) comme un nouveau partenaire d’interaction extracellulaire avec le PCSK9 et intracellulaire avec le complexe PCSK9-LDLR dans le réticulum endoplasmique des cellules HepG2 et Huh7. Nos études démontrent que GPC3 réduit l’activité extracellulaire de PCSK9 en agissant comme un compétiteur du LDLR pour la liaison avec PCSK9. Une meilleure compréhension des mécanismes de régulation et de dégradation du complexe PCKS9-LDLR permettra de mieux évaluer l’impact et l’efficacité des inhibiteurs de la protéine PCSK9.