Discovery of New Regulatory Proteins and Mechanisms in Muscle Biology and Disease
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In an effort to discover new regulators of muscle function, we identified a novel muscle-specific protein, Klhl40. Genetic deletion of Klhl40 in mice results in a nemaline myopathy-like phenotype with disruption of sarcomere function causing neonatal lethality. Nemaline myopathy (NM) typically results from sarcomere thin filament dysfunction, but the molecular function of Klhl40 is not known. We found that Klhl40 binds to two proteins: (1) nebulin (Neb), a sarcomere thin filament protein that is frequently mutated in NM; and (2) leiomodin 3 (Lmod3), a novel muscle-specific protein with putative thin filament actin polymerization activity. Klhl40 belongs to the BTB-BACK-Kelch (BBK) protein family, which typically promote protein ubiquitination and degradation, but we find that Klhl40 stabilizes its substrates. Thus, Neb and Lmod3 protein levels are diminished in Klhl40 deficient mice independent of any changes in their respective mRNA transcripts. Loss of KLHL40 in humans was recently reported to cause NM, and we find that NEB and LMOD3 are decreased in some KLHL40 mutant patients. However, the function of LMOD3 is also not known. To establish the role of LMOD3 in NM, we generated Lmod3 knockout mice by TALEN-mediated mutagenesis. Preliminary data shows that loss of Lmod3 results in a degenerative skeletal muscle myopathy. Thus, we propose that loss of Klhl40 directly results in decreased Neb and Lmod3 causing thin filament disruption and subsequent NM. In addition, we uncover the first BBK protein with a pro-stability function which has broad implications for future study of this protein family. In conjunction to our studies with Klh40, we found a closely neighboring gene in the antisense direction, Hhatl. Similar to Klhl40, we found that Hhatl expression is highly enriched in the heart and skeletal muscle although with notable expression in the central nervous system. Hhatl encodes for a putative membrane bound O-acyltransferase protein. Global deletion, but not heart or skeletal muscle-specific deletion, of Hhatl results in a failure to thrive phenotype with mid to late neonatal lethality. We outline future experiments to determine the nature and mechanism of the Hhatl knockout phenotype as well as possible means to delineate its function in striated muscles.