Department of Internal Medicine III, University of Heidelberg, Heidelberg, Germany.
Circ Res. 2012 Dec 7;111(12):1504-16. doi: 10.1161/CIRCRESAHA.112.271007. Epub 2012 Sep 12.
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.
We sought to determine whether molecules exist that control turnover of cardiac-specific proteins.
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.
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.
泛素-蛋白酶体系统在心肌细胞功能和稳态中的新作用表明蛋白质降解的严格调控是必要的。然而，关于该机制在心脏中的组成部分知之甚少。
我们试图确定是否存在控制心脏特异性蛋白周转的分子。
我们使用生物信息学方法来识别新的心脏丰富的肌节蛋白，鉴定出 F-box 和亮氨酸丰富重复蛋白 22（Fbxl22）。通过多种组织 Northern 和 Western Blot 分析以及人 cDNA 文库的定量逆转录聚合酶链反应证实了组织特异性表达。在新生和成年大鼠心室肌细胞以及鼠心组织中的免疫细胞化学定位实验将 Fbxl22 定位到肌节 Z 盘。为了检测心脏蛋白相互作用伙伴，我们使用 Fbxl22 作为诱饵进行酵母 2 杂交筛选。共免疫沉淀证实了 Fbxl22 与 S 期激酶相关蛋白 1 和 Cullin1、2（SCF（Skp1/Cul1/F-box）E3 连接酶的关键组成部分）的鉴定相互作用。此外，我们鉴定了几种潜在的底物，包括 Z 盘蛋白肌球蛋白重链结合蛋白 C 和原肌球蛋白 C。一致地，体外 Fbxl22 的过表达以剂量依赖性方式介导两种底物的降解，而用 MG-132 抑制蛋白酶体则明显减弱两种肌球蛋白重链结合蛋白 C 和原肌球蛋白 C 的降解。最后，在大鼠心肌细胞和斑马鱼胚胎中靶向敲低 Fbxl22 导致与严重受损的收缩功能和体内心肌病相关的肌球蛋白重链结合蛋白 C 的积累。
这些发现揭示了以前未被描述的心脏特异性 F 盒蛋白 Fbxl22 作为一种新型心脏 E3 连接酶的组成部分。Fbxl22 促进关键肌节蛋白，如肌球蛋白重链结合蛋白 C 和原肌球蛋白 C 的蛋白酶体依赖性降解，对于体内正常收缩功能的维持至关重要。
