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Rod mutations associated with MYH9-related disorders disrupt nonmuscle myosin-IIA assembly
Josef D Franke1, Fan Dong, Wayne L Rickoll
1Department of Biology, Developmental Cell and Molecular Biology Group, Duke University Medical Center, Durham, NC 27708-1000, USA.
Abstract:
MYH9-related disorders are autosomal dominant syndromes, variably affecting platelet formation, hearing, and kidney function, and result from mutations in the human nonmuscle myosin-IIA heavy chain gene. To understand the mechanisms by which mutations in the rod region disrupt nonmuscle myosin-IIA function, we examined the in vitro behavior of 4 common mutant forms of the rod (R1165C, D1424N, E1841K, and R1933Stop) compared with wild type. We used negative-stain electron microscopy to analyze paracrystal morphology, a model system for the assembly of individual myosin-II molecules into bipolar filaments. Wild-type tail fragments formed ordered paracrystal arrays, whereas mutants formed aberrant aggregates. In mixing experiments, the mutants act dominantly to interfere with the proper assembly of wild type. Using circular dichroism, we find that 2 mutants affect the alpha-helical coiled-coil structure of individual molecules, and 2 mutants disrupt the lateral associations among individual molecules necessary to form higher-order assemblies, helping explain the dominant effects of these mutants. These results demonstrate that the most common mutations in MYH9, lesions in the rod, cause defects in nonmuscle myosin-IIA assembly. Further, the application of these methods to biochemically characterize rod mutations could be extended to other myosins responsible for disease.
Insights
Mutations in the MYH9 gene cause disorders affecting platelets, hearing, and kidneys. These MYH9 rod mutations disrupt myosin-IIA assembly, explaining their dominant effects in patients.
Area of Science:
- Biochemistry
- Genetics
- Molecular Biology
Background:
- MYH9-related disorders are autosomal dominant conditions impacting platelet production, hearing, and kidney function.
- These syndromes arise from mutations in the gene encoding the heavy chain of human nonmuscle myosin-IIA (MYH9).
Purpose of the Study:
- To investigate how mutations in the MYH9 rod region affect nonmuscle myosin-IIA function.
- To elucidate the molecular mechanisms underlying the dominant inheritance pattern of MYH9-related disorders.
Main Methods:
- Analysis of four common MYH9 rod mutants (R1165C, D1424N, E1841K, R1933Stop) in vitro compared to wild type.
- Negative-stain electron microscopy to assess paracrystal morphology and filament assembly.
- Circular dichroism spectroscopy to evaluate molecular structure and interactions.
Main Results:
- Wild-type MYH9 tail fragments formed ordered paracrystals, while mutants aggregated aberrantly.
- Mutant MYH9 proteins dominantly interfered with wild-type assembly in mixing experiments.
- Specific mutants altered alpha-helical coiled-coil structure or disrupted lateral molecular associations, explaining dominant effects.
Conclusions:
- The most frequent MYH9 mutations, located in the rod domain, directly impair nonmuscle myosin-IIA assembly.
- These assembly defects are responsible for the dominant clinical manifestations of MYH9-related disorders.
- The employed biochemical methods can be applied to study other disease-related myosin mutations.
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