Spaich S, Will RD, Just S, Spaich S, Kuhn C, Frank D, Berger I, Wiemann S, Korn B, Koegl M, Backs J, Katus HA, Rottbauer W, Frey N

Rationale: The emerging role of the ubiquitin-proteasome system in cardiomyocyte function and homeostasis implies the necessity of tight regulation of protein degradation. Yet, 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: Utilizing ...

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a bioinformatic approach to identify novel cardiac-enriched sarcomere proteins we identified Fbxl22. Tissue-specific expression was confirmed by multiple tissue Northern and Western Blot analyses as well as qRT-PCR 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 two-hybrid screen employing Fbxl22 as bait. Coimmunoprecipitation confirmed the identified interactions of Fbxl22 with Skp1 and Cullin1, two 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, while 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 alpha-actinin and filamin C and is essential for maintenance of normal contractile function in vivo.

Date: Sep. 12, 2012

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