F-box and leucine-rich repeat protein 22 is a cardiac-enriched f-box protein that regulates sarcomeric protein turnover and is essential for maintenance of contractile function in vivo

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Abstract

RATIONALE: The emerging role of the ubiquitin-proteasome system in cardiomyocyte function and homeostasis implies the necessity of tight regulation of protein degradation. However, little is known about cardiac components of this machinery. OBJECTIVE: We sought to determine whether molecules exist that control turnover of cardiac-specific proteins. METHODS AND RESULTS: Using a bioinformatic approach to identify novel cardiac-enriched sarcomere proteins, we identified F-box and leucine-rich repeat protein 22 (Fbxl22). Tissue-specific expression was confirmed by multiple tissue Northern and Western Blot analyses as well as quantitative reverse-transcriptase polymerase chain reaction on a human cDNA library. Immunocolocalization experiments in neonatal and adult rat ventricular cardiomyocytes as well as murine heart tissue located Fbxl22 to the sarcomeric z-disc. To detect cardiac protein interaction partners, we performed a yeast 2-hybrid screen using Fbxl22 as bait. Coimmunoprecipitation confirmed the identified interactions of Fbxl22 with S-phase kinase-associated protein 1 and Cullin1, 2 critical components of SCF (Skp1/Cul1/F-box) E3-ligases. Moreover, we identified several potential substrates, including the z-disc proteins α-actinin and filamin C. Consistently, in vitro overexpression of Fbxl22-mediated degradation of both substrates in a dose-dependent fashion, whereas proteasome inhibition with MG-132 markedly attenuated degradation of both α-actinin and filamin C. Finally, targeted knockdown of Fbxl22 in rat cardiomyocytes as well as zebrafish embryos results in the accumulation of α-actinin associated with severely impaired contractile function and cardiomyopathy in vivo. CONCLUSIONS: These findings reveal the previously uncharacterized cardiac-specific F-box protein Fbxl22 as a component of a novel cardiac E3 ligase. Fbxl22 promotes the proteasome-dependent degradation of key sarcomeric proteins, such as α-actinin and filamin C, and is essential for maintenance of normal contractile function in vivo. © 2012 American Heart Association, Inc.

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Spaich, S., Will, R. D., Just, S., Spaich, S., Kuhn, C., Frank, D., … Frey, N. (2012). F-box and leucine-rich repeat protein 22 is a cardiac-enriched f-box protein that regulates sarcomeric protein turnover and is essential for maintenance of contractile function in vivo. Circulation Research, 111(12), 1504–1516. https://doi.org/10.1161/CIRCRESAHA.112.271007

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