Identification of a motif in BMRP required for interaction with Bcl‐2 by site‐directed mutagenesis studies

Abstract

Bcl-2 is an anti-apoptotic protein that inhibits apoptosis elicited by multiple stimuli in a large variety of cell types. BMRP (also known as MRPL41) was identified as a Bcl-2 binding protein and shown to promote apoptosis. Previous studies indicated that the amino-terminal two-thirds of BMRP contain the domain(s) required for its interaction with Bcl-2, and that this region of the protein is responsible for the majority of the apoptosis-inducing activity of BMRP. We have performed site-directed mutagenesis analyses to further characterize the BMRP/Bcl-2 interaction and the pro-apoptotic activity of BMRP. The results obtained indicate that the 13-17 amino acid region of BMRP is necessary for its binding to Bcl-2. Further mutagenesis of this motif shows that amino acid residue aspartic acid (D) 16 of BMRP is essential for the BMRP/Bcl-2 interaction. Functional analyses conducted in mammalian cells with BMRP site-directed mutants BMRP(13Ala17) and BMRP(D16A) indicate that these mutants induce apoptosis through a caspase-mediated pathway, and that they kill cells slightly more potently than wild-type BMRP. Bcl-2 is still able to counteract BMRP(D16A)-induced cell death significantly, but not as completely as when tested against wild-type BMRP. These results suggest that the apoptosis-inducing ability of wild-type BMRP is blocked by Bcl-2 through several mechanisms.

Genome-Wide Prediction and Validation of Peptides That Bind Human Prosurvival Bcl-2 Proteins

“…MRPL41 (also known as BMRP) is an apoptosis-inducing mitochondrial protein that interacts with Bcl-2. Mutation of Asp16 to Ala in full-length MRPL41 abolished Bcl-2 binding in a two-hybrid assay [36]. This residue corresponds to the conserved aspartate in our predicted BH3 motif, which as a peptide binds Bcl-2 with a K D of 190 nM.…”

Structure and Function of the Mitochondrial Ribosome

“…It forms tight interactions with many surrounding mitoribosomal proteins, as well as with the 16S rRNA, and it is unlikely to be easily dissociated from the ribosomal particle. The putative Bcl-2 interaction sites near the mL41 N terminus (134) are either buried in the 16S rRNA and are, therefore, inaccessible in the conformation observed in our structure or are missing in the mature protein due to cleavage of the mitochondrial targeting sequence. Therefore, binding to Bcl-2 and any apoptotic activity of mL41 likely take place in the cytosol (134) rather than after mitochondrial import and incorporation into the mitoribosome.…”

“…Bcl-2 proteins interact directly with mL41 (133) as a means of attenuating its apoptotic activity (134). In the structure of the 39S subunit, mL41 assumes a mostly extended conformation (Figure 6d ) (60).…”

Mitochondrial ribosomes in cancer

“…Most probably, this interaction/s do not occur with mL41 embedded within the mt-LSU structure [47, 49]. In fact, the BCL-2 binding sites have been found near the N-terminus of mL41 [103, 104]. They correspond to motifs that are either absent in the mature protein owing to the cleavage of the mitochondrial targeting sequence after its import into mitochondria, or might be buried within the 16S rRNA - thereby making it inaccessible for interaction with BCL-2.…”

“…They correspond to motifs that are either absent in the mature protein owing to the cleavage of the mitochondrial targeting sequence after its import into mitochondria, or might be buried within the 16S rRNA - thereby making it inaccessible for interaction with BCL-2. Therefore, the interaction of mL41 with BCL-2 and its apoptotic activity are likely to occur in the cytosol before its import into mitochondrial and incorporation in mitoribosomes [10, 104]. Further studies are needed to unravel the mechanisms by which the mL41 precursor induces apoptosis, and those that control mL41 processing in non-malignant and cancer cells.…”