Browsing by Subject "Protein aggregation"

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    A biochemical and biophysical study of dystrophin.
    (2011-07) Henderson, Davin Michael
    The primary role of a muscle cell is to contract and produce force that moves an organism. A vast majority of a muscle is made of the proteins in contraction machinery and nearly all energy utilized by the cell is consumed in this process. However, an equally important but substantially smaller portion of the muscle cell is dedicated to the preservation of cell membrane integrity. The costamere is an elaborate matrix of cytoskeleton associated and transmembrane proteins that form a support lattice between the plasma membrane and contractile apparatus. The dystrophin glycoprotein complex (DGC) is a structurally important member of the costamere and has been shown to link microtubules, thin and intermediate filaments of the cytoskeleton with major components of the extracellular matrix. In the DGC, dystrophin is responsible for attachments with intracellular cytoskeletal components and the transmembrane protein dystroglycan. One of the most common diseases afflicting muscle is Duchenne muscular dystrophy (DMD), which is caused by mutations in the gene encoding the protein dystrophin. The focus of my thesis is to better understand the biochemical and biophysical properties of dystrophin. Specifically, I investigated the actin binding properties of dystrophin in the context of its functional domains as well as the consequences of disease causing missense mutations localized to actin-binding domain 1 (ABD1). Additionally, I characterized the biophysical properties of internally truncated dystrophin proteins under development for treatment of DMD. It has been twenty years since dystrophin was hypothesized to bind actin and even today we are learning more about this fascinating interaction. My thesis expands our understanding of the dystrophin-actin interaction in three ways. First, I showed that full-length dystrophin interacts with the actin isoforms expressed in muscle with equivalent affinities. Second, I showed that the thermally stable C-terminal domain of dystrophin is required for full actin binding activity. Third, in collaboration with the Thomas lab, we showed that dystrophin and utrophin uniquely alter the physical properties of actin filaments. Disease causing missense mutations in the dystrophin gene are scattered in many functional domains but we chose to study a cluster localized to ABD1 with hope that we would find amino acids important for actin binding. We hypothesized that mutations in ABD1 would disrupt actin binding and therefore lead to disease though loss of an essential interacting partner. However, no mutation dramatically disrupted actin binding but instead lead to loss of thermal stability and protein aggregation. My thesis work was the first to show evidence that protein stability and aggregation may play a role in the pathogenesis of dystrophinopathies. DMD currently has no effective treatment but many promising therapies are being pursued. Many laboratories are pursuing therapies for DMD and multiple techniques are being pursued including adeno-associated viral gene therapy, protein replacement therapy, exon skipping therapy and stop codon read though therapy. For gene therapy and protein therapy, the size of the dystrophin or utrophin coding sequence has been reduced by deletion of internal domains, which retains important N- and C-terminal ligand binding sites. I set out to test the stability of internally deleted therapy proteins to ensure that no unwanted structural perturbations were caused by internal deletion. Additionally, I tested a set of N-and C-terminal truncations of dystrophin and a dystrophin related protein, utrophin for comparison to internally deleted versions of these proteins used in therapy. I found that the thermal stability of utrophin was uniform from N- to C-terminus and that internal deletion did not affect protein stability. I also found that the N-terminal half of dystrophin had a lower thermal stability compared to the C-terminal half and, to our surprise, internally deleted dystrophin proteins showed marked thermal instability and aggregation.
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    The Dual Dependency of Varying Liposomes and Protein on Available αS Conformers
    (2016) Peterson, Kathryn
    α-Synuclein (αS) is a protein commonly found in protein aggregates associated with Parkinson’s disease (PD). This intrinsically disordered protein is known to regulate synaptic vesicle (SV) trafficking in the pre-synaptic clefts of most neurons. Improper trafficking of SVs results in miscommunication between neurons, which could lead to symptoms of PD such as muscular tremors. Despite the prevalence of PD, much is still left unknown about the mechanism that causes protein aggregation due to αS binding to SVs. SVs are unique membranes as a result of their high cholesterol content (45%) and small diameter (0.03 microns). Both factors induce strain in the membrane leading to high fusion potential of the SV. For this research two SV mimics (simple and complex) were designed, utilizing a mass spectrometry study on SV membrane composition.1 The simple SV mimic measured the effects of interacting head groups and cholesterol on membrane annealing in the presence of αS using a Carboxyfluorescein (CF) release assay .To further probe the question of conformational changes of αS in the presence of membrane Circular Dichroism (CD) monitored the secondary structure character. Both membrane annealing and a change in secondary structure were observed making it necessary to further investigate the relationship between protein and membrane with other methods. Using Differential Scanning Calorimetry (DSC) to monitor a lipid transition I hypothesized that αS has specificity for high curvature and complex composition of membrane. We tested this by varying liposome sizes and cholesterol content. Oppositely, the membranes impact on αS conformers was studied utilizing a DSC protein transition to see its effects. A conformational shift was found in the presence of complex SV mimic, showing αS’s conformational specificity for this highly complex mimic. Due to this preference, a binding mechanism using the complex SV mimic needs to be studied. The mechanism of αS binding to membrane has the potential to shed light on the pathogenesis of αS in amyloid formation. Through Isothermal Titration Calorimetry (ITC) we will predict a simulated binding model for αS that will give more information about possible reasons for protein aggregation and benefit future studies on PD.