Abstract 991: The Novel BTB/POZ Protein, RhoBTB3, Regulates Mitochondrial Function Via The Ubiquitin Proteasome System
Although BTB/POZ family proteins play a critical role in the development of various organs, and are recently known as a substrate adaptor of CUL3 ubiquitin ligases, the involvement of these proteins in heart development remains unknown. We recently isolated the novel protein, RhoBTB3, an 83-kD protein which comprises an N-terminal Rho GTPase resembling domain and two tandem BTB domains. The purpose of this study was to investigate the molecular and functional characteristics of this protein in mice heart.
[Methods and Results]
RhoBTB3 was faintly expressed in the embryonic heart, but was strongly expressed in the adult heart. It was weakly expressed in adult brain and testis.
RhoBTB3-GFP fusion protein cDNA was transfected into primary cultured rat cardiomyocytes using adenovirus. Fluorescence microscopy revealed that RhoBTB3 was located at the cytoplasm of living cardiomyocytes.
HA-tagged RhoBTB3 and MYC-tagged CUL family proteins (1 – 5) were co-transfected into COS7 cells. RhoBTB3 was co-immunoprecipitated with CUL3 in vitro, but not with other CUL proteins.
The various mutant RhoBTB3 and wild type CUL3 protein were examined by immunoprecipitation assay. Both the first BTB domain and second BTB domain can bind to CUL3 protein independently.
It was difficult to detect RhoBTB3 in protein analysis. To determine whether or not RhoBTB3 itself could be the Cul3 ubiquitin target, RhoBTB3 protein was measured under the presence of proteasome inhibitor MG132 and protein synthesis inhibitor cycloheximide. The RhoBTB3 protein was immediately degraded and dropped below the detection level within 2 hours.
The transgenic mice which overexpressed the heart-restricted RhoBTB3 were healthy at a young age.
But after 3 months, they gradually showed heart failure and finally fell into sudden death. Interestingly before heart failure, electron microscopy demonstrated that mitochondria dramatically increased in number and showed deformity in RhoBTB3 transgenic hearts.
[Conclusions] RhoBTB3 is mainly expressed in the adult heart, and its protein was rapidly degraded with the CUL3-mediated ubiquitin proteasome system in the heart. The overexpression of RhoBTB3 caused a marked increase in abnormal mitochondria, followed by severe heart failure.