Molecular and Cellular Forces involved in the Pathogenesis and Therapy of Muscular Dystrophy

Abstract

Dystrophin is an essential muscle protein, which protects the cell membrane from damage. Utrophin is a dystrophin homologue with very similar functions. Duchenne muscular dystrophy (DMD) is a lethal muscle wasting disease caused by the absence of dystrophin, and utrophin has been proposed as replacement to treat DMD. Muscle is a highly mechanical organ and dystrophin and utrophin contribute to its function. In this thesis I characterized the mechanical contribution of dystrophin and disease-causing mutants at the cellular level, and the effect of expression system and phosphorylation on dystrophin’s and utrophin’s mechanical stiffness at the molecular level. Expression of dystrophin in muscle cells increased vinculin tension, while expression of dystrophin mutants did not. Activation levels of mechanosensitive protein, YAP and ERK1, were also higher for cells expressing wild type dystrophin, compared to the cell expressing mutant dystrophin which also showed defective migration phenotypes. These data demonstrate that dystrophin is an allosteric regulator of vinculin tension, dystrophin mutants impaired mechanical function, and that the migration and mechanotransduction processes of cells expressing disease-associated dystrophin mutants are altered. Eukaryotes and bacteria modify their proteins differently, which can impact their functional properties. We expressed the same utrophin fragment using bacteria, insect cells, and mammalian cells and demonstrated eukaryotic cells phosphorylate the fragment while bacteria did not. We probed the conformational stability for the construct and showed the expression system modified the protein stiffness, and our data indicates phosphorylation is responsible for the increase in mechanical stability. Thus, utrophin stiffness can be tuned by altering phosphorylation sites.