Abstract:
BNip3 resembles Bcl-2-related proteins, based on sequence similarity to the Bcl-2 homology 3 (BH3) domain and COON-terminal transmembrane (TM) domain. Characteristically, the 'BH3-only' class of Bcl-2 proteins heterodimerize with Bcl-2/Bcl-xL and induce cell death through their BH3 domain. BNip3 and its 'C. elegans' orthologue, CeBNip3 interact with cell death repressors Bcl-2, Bcl-xL and CED-9 in yeast and mammalian expression systems. Unlike other BH3-only agonists, deletion mapping of BNip3 excluded its putative BH3 domain and identified the NH 2-terminus and TM domain critical for Bcl-2 heterodimerization, and either region sufficient for Bcl-xL interaction. Similar mapping studies of CeBNip3 demonstrated its association with CED-9 and Bcl-xL exclusively through its TM domain. Both BNip3 and CeBNip3 induce cell death in several cell lines. Yet, the removal of the BH3-like domain in either protein does not diminish its cytotoxicity. Some Bcl-2 homologues form homodimers through regions of shared homologyto regulate their functio . Following 'in vitro' transcription/translation, BNip3 was expressed primarily as a 60 kDa protein along with a 30 kDa product. Interaction of BNip3 monomers was observed in the yeast two-hybrid system and deletion mapping localized the region mediating homodimerization to the TM domain. The BNip3 TM domain is also critical for mitochondria) targeting and cell death function. Its removal results in cytosolic expression and eliminates both its homologous interaction and cytotoxicity. Several BNip3 TM domain mutations were found to disrupt SDS-resistant BNip3 homodimerization, but did not interfere with its mitochondria) localization or killing activity. The substitution of the BNip3 TM domain with cytochrome ' b5' targeting sequence directed protein expression to nonmitochondrial sites, promoting both cell death and Bcl-2/Bcl-xL heterodimerization. Therefore, the function of BNip3 is independent of homodimerization, but the TM domain is necessary for mitochondria) targeting and cell death function. In conclusion, BNip3 is a mitochondria) cell death-inducing protein, which although structurally related to the Bcl-2 family appears to heterodimerize and to induce cell death in the absence of its BH3-like domain. Thus, BNip3-related proteins define a new class of cell death proteins, which interact with Bcl-2, Bcl-xL and CED-9 and kill independent of a BH3 domain.
