Characterization of the effects of steroid-resistant nephrotic syndrome associated MYO1E mutations on myosin 1e activity and podocyte functions

Abstract

MYO1E mutations are associated with familial pediatric nephrotic syndrome, a disease in which pharmaceutical treatment is limited to immunosuppressive drugs such as steroids and cyclosporine but still often fails to prevent progression to end-stage renal disease. While Myo1e deficiency leads to glomerular filtration abnormality, including podocyte foot process effacement and glomerular basement membrane thickening, developing a precise treatment for this disease is hindered, as the underlying mechanisms linking MYO1E mutations and nephrotic syndrome are unclear. With the power of whole genome and exome sequencing, multiple novel Myo1e variants have been rapidly identified from SRNS cohorts. To determine whether a MYO1E mutation identified in patients is likely to be pathogenic or benign, we have characterized the functional effects of novel sequence variants in cultured podocytes. Differential protein degradation, localization, endocytic and motor activities of Myo1eT119I and Myo1eD388H have been discovered (Chapter 2). Specifically, even when expressed as a full-length protein, Myo1eT119I is deficient in localization to podocyte junctions and clathrin-coated vesicles (CCVs). Consistent with the hypothesis that Myo1eT119I is a loss-of-function mutant, cells expressing Myo1eT119I exhibit decreased CCV density and prolonged CCV lifetimes. The junctional and CCV localization of Myo1eD388H is not affected but it exhibits increased association with structures in the membrane-actin interface. Most importantly, Myo1eD388H is deficient in ATP hydrolysis and actin filament translocation. We have also characterized other MYO1E variants to provide cell-based evidence to assist in the curation of variants of uncertain significance (Chapter 3). Unexpectedly, while Myo1eD185G may be considered as a likely benign variant based on the population and computational predictions, it exhibits prolonged association with the podocyte junctions, while no junctional localization and dissociation abnormality was found in a likely deleterious variant, Myo1eR523W. We also discovered that localization of Myo1edel3094-7 to the podocyte junctions can be partially restored with a proteasomal inhibitor treatment, which may be considered as a potential treatment for patients with this variant. Finally, to follow up on the questions derived from Chapter 2, we examined our hypothesis that Myo1eD388H tail is constitutively active, we demonstrated the similar junctional exchange but differential endocytic activity of Myo1eD388H and Myo1eTAIL, revealing the critical regulation of Myo1e motor to tail domain when it comes to clathrin-mediated endocytosis (Appendix 1). Overall, the studies documented here have uncovered the differential molecular defects of steroid resistant nephrotic syndrome (SRNS)-associated Myo1e variants and further elucidated the underlying mechanisms of podocyte disease.