Divergent Molecular Effects of Desmin Mutations on Protein Assembly in Myofibrillar Myopathy

Abstract

Mutations in the intermediate filament protein desmin cause a distinct class of myofibrillar myopathies that are characterized by deposition of aggregated desmin. To assess the effect of different disease-associated mutations at the molecular level, we applied confocal single-particle fluorescence spectroscopy. We studied the de novo aggregation properties of desmin in vitro and the aggregation state of desmin in homogenates of transfected cells rendering purification unnecessary. We detected divergent assembly patterns for 3 different desmin missense mutations. R350P-desmin showed a strong inhibition of assembly formation that was associated with a reduced level of tetramers and an increase in dimers in native cell extracts. E413K-desmin formed hyperstable tetramers. For R454W-desmin, there were subtle effects on assembly at the dimer and tetramer levels by single-particle spectroscopy that are not detectable by classical fluorescence microscopy. We also found that R350P-desmin efficiently interacts with the wild-type protein resulting in a dominant-negative effect on desmin assembly. Taken together, these results provide a molecular basis for a detailed functional classification of mutations in the desmin gene. The findings may also have implications for diagnostic and therapeutic strategies for primary desminopathies based on the different molecular events that disrupt physiological filament formation.

Dual Color Photoactivation Localization Microscopy of Cardiomyopathy-associated Desmin Mutants

“…Different groups investigated the homozygous expression of p.R454W desmin in different cell lines, leading to conflicting results concerning the degree of aggregate formation (28,44). In our hands, homozygously transfected p.R454W desmin formed filament networks independently of the cell type investigated.…”

“…However, in that study, co-localization of wild-type and mutant desmin could not be analyzed for technical reasons. The amount of dimers, tetramers, and oligomers of p.R454W in transfected cells was previously determined by single particle fluorescence spectroscopy, demonstrating that p.R454W differs only marginally from the wild type (28). Consequently, the pathogenic role of this variant is not clear.…”

“…Because we used tagged desmin constructs carrying fluorescent proteins at the C terminus, we and others (28) excluded an influence of the tag on filament or aggregate formation by immunolabeling (see Fig. 2C).…”

Myofibrillar myopathies

“…The Arg350Pro and Glu413Lys mutants form aggregates in HEK (human embryonal kidney) 293 cells that lack endogenous desmin, and Arg454Trp-mutant forms a filamentous network like wild-type desmin. Spectroscopy shows that the Arg350Pro mutant inhibits desmin assembly, the Glu413Lys mutant forms hyperstable tetramers, and the Arg454Trp mutant shows only a subtle defect in filament assembly [14]. However, another expression system using MEF Vim −/− (Vimentin knockout mouse embryo fibroblast) cells shows the Arg454Trp mutant forms abnormally short filamentous structures and only forms a filamentous network when cotransfected with wild-type desmin [15].…”

Arrhythmogenic cardiomyopathies (ACs): diagnosis, risk stratification and management

“…DES mutations lead to heterogeneous myopathies ranging from skeletal muscle disease to isolated cardiomyopathy 24. So far, less than 10 pathogenic variants linked to AC have been identified in DES gene.…”