No description available.

No description available.

Apoptosis is essential for development and the maintenance of cellular homeostasis and is frequently dysregulated in disease states. Proteins of the BCL-2 family are key modulators of this process and are thus ideal therapeutic targets. In response to diverse apoptotic stimuli, the pro-apoptotic member of BCL-2 family, BAX, redistributes from the cytosol to the mitochondria or endoplasmic reticulum and primes cells for death. The structural changes that enable this lethal protein to transition from a cytosolic form to a membrane-bound form remain poorly understood. Elucidating this process is a necessary step in the development of BAX as a novel therapeutic target for the treatment of cancer, as well as autoimmune and neurodegenerative disorders. A three-part study, utilizing computational modeling and biological assays, was used to examine how BAX, and similar proteins, transition to membranes. The first part tested the hypothesis that the C-terminal α9 helix regulates the distribution and activity of BAX by functioning as a "molecular switch" to trigger conformational changes that enable the protein to redistribute from the cytosol to mitochondrial membrane. Computational analysis, tested in biological assays, revealed a new finding: that the α9 helix can dock into a hydrophobic groove of BAX in two opposite directions – in a self-associated, forward orientation and a previously, unknown reverse orientation that enables dimerization and apoptosis. Peptides, made to mimic the α9-helix, were able to induce the mitochondrial translocation of BAX, but not when key residues in the hydrophobic groove were mutated. Such findings indicate that the α9 helix of BAX can function as a "molecular switch" to mediate occupancy of the hydrophobic groove and regulate the membrane-binding activity of BAX. This new discovery contributes to the understanding of how BAX functions during apoptosis and can lead to the design of new therapeutic approaches based on manipulating the occupancy of the hydrophobic groove. The second and third parts of the study used computational modeling to examine how the helical stability of proteins relates to their ability to functionally transition. Analysis of BAX, as a prototypical transitioning protein, revealed that it has a broad variation in the distribution of its helical interaction energy. This observation led to the hypothesis tested, that proteins which undergo 3D structural transitions during execution of their function have broad variations in the distribution of their helical interaction energies. The result of this study, after examination of a large group of all-alpha proteins, was the development of a novel, predictive computational method, based on measuring helical interactions energies, which can be used to identify new proteins that undergo structural transitioning in the execution of their function. When this method was used to examine transitioning in other members the BCL-2 family, a strong agreement with the published experimental findings resulted. Further, it was revealed that the binding of a ligand, such as a small peptide, to a protein can have significant stabilizing or destabilizing influences that impact upon the activation and function of the protein. This computational analysis thus contributes to a better understanding of the function and regulation of the BCL-2 family members and also offers the means by which peptide mimics that modulate protein activity can be designed for testing in therapeutic endeavors.

Liposomal delivery systems hold considerable promise for improvement of cancer therapy provided that critical formulation design criteria can be met. The main objective of the current project was to enable quality by design in the formulation of liposomal delivery systems by developing comprehensive, mechanism-based mathematical models of drug loading, binding and release kinetics that take into account not only the therapeutic requirement but the physicochemical properties of the drug, the bilayer membrane, and the intraliposomal microenvironment. Membrane binding of the drug affects both drug loading and release from liposomes. The influence of bilayer composition and phase structure on the partitioning behavior of a model non-polar drug, dexamethasone, and its water soluble prodrug, dexamethasone phosphate, was evaluated. Consequently, a quantitative dependence of the partition coefficient on the free surface area of the bilayer, a property related to acyl chain ordering, was noted. The efficacy of liposomal formulations is critically dependent on the drug release rates from liposomes. However, various formulation efforts to design optimal release rates are futile without a validated characterization method. The pitfalls of the commonly used dynamic dialysis method for determination of apparent release kinetics from nanoparticles were highlighted along with the experimental and mathematical approaches to overcome them. The value of using mechanism-based models to obtain the actual rate constant for nanoparticle release was demonstrated. A novel method to improve liposomal loading of poorly soluble ionizable drugs using supersaturated drug solutions was developed using the model drug AR-67 (7-t-butyldimethylsilyl-10-hydroxycamptothecin), a poorly soluble camptothecin analogue. Enhanced loading with a drug to lipid ratio of 0.17 was achieved and the rate and extent of loading was explained by a mathematical model that took into account the chemical equilibria inside and outside the vesicles and the transport kinetics of various permeable species across the lipid bilayer and the dialysis membrane. Tunable liposomal release kinetics would be highly desirable to meet the varying therapeutic requirements. A large range of liposome release half-lives from 1 hr to 892 hr were obtained by modulation of intraliposomal pH and lipid composition using dexamethasone phosphate as a model ionizable drug. The mathematical models developed were successful in accounting for the change in apparent permeability with change in intraliposomal pH and bilayer free surface area. This work demonstrates the critical role of mechanism-based models in design of liposomal formulations.

Lipoxygenasen sind nichthämeisenhaltige Dioxygenasen, die die Bildung von Hydroperoxiden aus molekularem Sauerstoff und mehrfach ungesättigten Fettsäuren katalysieren. Im Rahmen dieser Arbeit wurden verschiedene Lipoxygenase-Ligand-Wechselwirkungen untersucht: a) Durch gezielte Substratveränderungen und ortsgerichtete Mutagenese konnte gezeigt werden, dass Fettsäuren normalerweise mit dem Methylende in der Bindungstasche tierischer Lipoxygenasen gebunden werden. Unterschiedliche Positionsspezifitäten basieren demzufolge auf dem Volumen der Substratbindungstasche (Volumenhypothese). Darüber hinaus konnte bei der 15-Lipoxygenase eine inverse Substratbindung (Carboxylgruppe im aktiven Zentrum) durch Modifikation beider Enden der Fettsäure erzwungen werden, wodurch ausschließlich 5-Lipoxygenierung katalysiert wurde. b) Untersuchungen mit dem Hemmstoff Ebselen ergaben unterschiedliche Hemmmechanismen für verschiedene Enzymzustände. Die Hemmung der Lipoxygenase im Grundzustand (Fe[II]) erfolgt durch kovalente Bindung und Veränderung der Eisenligandensphäre irreversibel nach einem nicht-kompetitiven Mechanismus. Dagegen wird die aktive Lipoxygenase (Fe[III]) nur noch kompetitiv durch Ebselen gehemmt. c) Die Membranbindung der tierischen 15-Lipoxygenase erfolgt über hydrophobe Wechselwirkungen, vermittelt durch oberflächenexponierte, hydrophobe Aminosäuren aus beiden Enzymdomänen. Die Expression einer enzymatisch aktiven Trunkationsmutante, der die N-terminale Domäne fehlt, zeigte, dass diese nicht essentiell ist für die Membranbindung. / Lipoxygenases are nonheme iron-containig dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids to hydroperoxy derivatives. Here, the interaction of lipoxygenases with various ligands was investigated: a) From substrate modifications and site directed mutagenesis it was concluded that fatty acids are bound with their methyl end in the substrate binding pocket of mammalian lipoxygenases. The positional specificity is therefore related to different volumes of this binding pocket (space-based hypothesis). For the 15-lipoxygenase an inverse substrate binding (carboxy terminus in the pocket) could be forced by simultaneous modification of both ends of the fatty acid. This lead to an exclusive 5-lipoxygenation by the 15-lipoxygenase. b) The mechanism of lipoxygenase inhibition by ebselen depends on the enzyme's state. The groundstate lipoxygenase (containing Fe[II]) is irreversibly inhibited in a non-competetive manner due to covalent modification and alteration of the iron ligand sphere. The active enzyme (containing Fe[III]) on the other hand is only competetively inhibited. c) The membrane binding of the mammalian 15-lipoxygenase is based on hydrophobic interactions mediated by solvent exposed, hydrophobic amino acid residues of both enzyme domains. The expression of an enzymatically active truncation mutant, which lacks the entire N-terminal domain, showed that this domain is not essential for membrane binding.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

No description available.

Symmetric cell division in gram-negative bacteria is essential for generating two equal-sized daughter cells, each containing cellular material crucial for growth and future replication. The Min system, comprised of proteins MinC, MinD and MinE, is particularly important for this process since its deletion leads to minicells incapable of further replication. This thesis focuses on the interactions involving MinE that are important for allowing cell division at the mid-cell and for directing the dynamic localization of MinD that is observed in vivo. Previous experiments have shown that the MinE protein contains an N-terminal region that is required to stimulate MinD-catalyzed ATP hydrolysis in the Min protein interaction cycle. However, MinD-binding residues in MinE identified by in vitro MinD ATPase assays were subsequently found to be buried in the hydrophobic dimeric interface in the MinE structure, raising the possibility that these residues are not directly involved in the interaction. To address this issue, the ability of N-terminal MinE peptides to stimulate MinD activity was studied to determine the role of these residues in MinD activation. Our results implied that MinE likely undergoes a change in conformation or oligomerization state before binding MinD. In addition we performed circular dichroism spectroscopy of MinE. The data suggest that direct interactions between MinE and the lipid membrane can lead to conformational changes in MinE. Using NMR spectroscopy in an attempt to observe this structure change, different membrane-mimetic environments were tested. However the results strongly suggest that structural studies on the membrane-bound state of MinE will pose significant challenges. Taken together, the results in this thesis open the door for further exploration of the interactions involving MinE in order to gain a better understanding of the dynamic localization patterns formed by these proteins in vivo.

In order to associate with the cytoplasmic leaflet of the plasma membrane, many cytosolic signalling proteins possess a distinct lipid binding domain as part of their overall fold. Here, a multiscale simulation approach has been used to investigate three membrane-binding proteins involved in cellular processes such as growth and proliferation. The pleckstrin homology (PH) domain from the general receptor for phosphoinositides 1 (GRP1-PH) binds phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P₃) with high affinity and specificity. To investigate how this peripheral protein is able to locate its target lipid in the complex membrane environment, Brownian dynamics (BD) simulations were employed to explore association pathways for GRP1-PH binding to PI(3,4,5)P₃ embedded in membranes with different surface charge densities and distributions. The results indicated that non-PI(3,4,5)P₃ lipids can act as decoys to disrupt PI(3,4,5)P₃ binding, but that at approximately physiological anionic lipid concentrations steering towards PI(3,4,5)P₃ is actually enhanced. Atomistic molecular dynamics (MD) simulations revealed substantial membrane penetration of membrane-bound GRP1-PH, evident when non-equilibrium, steered MD simulations were used to forcibly dissociate the protein from the membrane surface. Atomistic and coarse grained (CG) MD simulations of the phosphatase and tensin homologue deleted on chromosome ten (PTEN) tumour suppressor, which also binds PI(3,4,5)P₃, detected numerous non-specific protein-lipid contacts and anionic lipid clustering around PTEN that can be modulated by selective in silico mutagenesis. These results suggested a dual recognition model of membrane binding, with non-specific membrane interactions complementing the protein-ligand interaction. Molecular docking and MD simulations were used to characterise the lipid binding properties of kindlin-1 PH. Simulations demonstrated that a dynamic salt bridge was responsible for controlling the accessibility of the binding site. Electrostatics calculations applied to a variety of PH domains suggested that their molecular dipole moments are typically aligned with their ligand binding sites, which has implications for steering and ligand electrostatic funnelling.