A biochemical and biophysical study of dystrophin.

Abstract

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.