The ubiquitin-proteasome system (UPS) is critical for specific degradation of cellular proteins and plays a pivotal role on protein breakdown in muscle atrophy. Here, we show that ZNF216 directly binds polyubiquitin chains through its N-terminal A20-type zinc-finger domain and associates with the 26S proteasome. ZNF216 was colocalized with the aggresome, which contains ubiquitinylated proteins and other UPS components. Expression of Znf216 was increased in both denervation-and fasting-induced muscle atrophy and upregulated by expression of constitutively active FOXO, a master regulator of muscle atrophy. Mice deficient in Znf216 exhibited resistance to denervation-induced atrophy, and ubiquitinylated proteins markedly accumulated in neurectomized muscle compared to wild-type mice. These data suggest that ZNF216 functions in protein degradation via the UPS and plays a crucial role in muscle atrophy.
Rationale: A homozygous disruption or genetic mutation of the bag3 gene, a member of the Bcl-2-associated athanogene (BAG) family proteins, causes cardiomyopathy and myofibrillar myopathy that is characterized by myofibril and Z-disc disruption. However, the detailed disease mechanism is not yet fully understood.
Objective: bag3؊/؊ mice exhibit differences in the extent of muscle degeneration between muscle groups with muscles experiencing the most usage degenerating at an accelerated rate. Usage-dependent muscle degeneration suggests a role for BAG3 in supporting cytoskeletal connections between the Z-disc and myofibrils under mechanical stress. The mechanism by which myofibrillar structure is maintained under mechanical stress remains unclear. The purpose of the study is to clarify the detailed molecular mechanism of BAG3-mediated muscle maintenance under mechanical stress.
Methods and Results:
BAG3 protein binds to and regulates Hsp70 chaperone activity. The BAG3 protein contains a WW domain and a prolinerich region with SH3-binding motifs, suggesting that it may interact with proteins relevant to signal transduction, recruiting Hsp70 to signaling complexes and altering cell responses. BAG3 overexpression has been observed in human cancers. We show here that homozygous BAG3-deficient mouse embryonic fibroblasts (MEF) exhibit delayed formation of filopodia and focal adhesion complexes when freshly plated. BAG3-deficient MEFs show reduced cell motility in culture. We observed that endogenous BAG3 protein is highly expressed in many human epithelial cancer cell lines, especially adenocarcinomas. Gene transfer-mediated overexpression of BAG3 increased motility of Cos7 cell and several human cancer cell lines, including breast cancer MCF7 and prostate cancer DU145 and ALVA31 cell lines. Conversely, reduction of BAG3 protein by RNA interference (RNAi) decreased cell motility in four of four epithelial tumor lines tested. We observed an influence of BAG3 on cell adhesion in culture. In Cos7 kidney epithelial cells, BAG3 protein partially colocalizes with actin at the leading edge of migrating cells, wherein active actin polymerization and nucleation occur. RNAi-mediated reductions in BAG3 expression were associated with decreased Rac1 activity, suggesting a role for BAG3 in regulating this small GTPase involved in actin-cytoskeleton dynamics. In mice, RNAi-mediated reductions in BAG3 in a human tumor xenograft suppressed invasion and metastasis in vivo. Thus, the high levels of BAG3 protein seen in some epithelial cancer cell lines may be relevant to mechanisms of tumor invasion and metastasis.
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