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

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. J. Cell. Biochem. 113: 3498–3508, 2012. © 2012 Wiley Periodicals, Inc.     

Tetraspanins stimulate protein synthesis in myeloma cell lines

Intensive protein synthesis is a unique and differential trait of multiple myeloma (MM) cells. Previously we showed that tetraspanin (CD81, CD82) overexpression in MM cell lines attenuated Akt/mTOR cascades, activated UPR, and caused autophagic death, suggesting breach of protein homeostasis. Here, we explored the role of protein synthesis in the tetraspanin-induced MM cell death. Contrary to attenuation of the major metabolic regulator, mTOR we determined elevated steady-state levels of protein in CD81N1/CD82N1 transfected MM lines (RPMI-8226, CAG). Elevated levels of immunoglobulins supported increased protein production in RPMI-8226. Changes in cell morphology consistent with elevated protein synthesis were also determined (cell, nuclei, and nucleoli sizes and ratios). Increased levels of phospho-rpS6 and decreased levels of phospho-AMPK were consistent with increased translation but independent of mTOR. Involvement of p38 and its role in tetraspanin induced translation and cell death were demonstrated. Microarray analyses of tetraspanin transfected MM cell lines revealed activation of protein synthesis signaling cascades and signals implicated in ribosome biogenesis (snoRNAs). Finally, we showed tetraspanins elevated protein synthesis was instrumental to MM cells' death. This work explores and demonstrates that excessive protein translation can be detrimental to MM cell lines and therefore may present a therapeutic target. Proteostasis is particularly important in MM because it integrates the high levels of protein production unique to myeloma cells with critically important microenvironmental cues. We suggest that increasing translation may be the path of least resistance in MM and thus may afford a novel platform for strategically designed therapy.