||Background: Bcl2L12 is a new member of Bcl2 family, which is reported to harbor BH3-like domain with an unidentified function. Previous study showed that Bcl2L12 involved in regulation of post-mitochondrial apoptotic events through multiple molecular mechanisms including controls the transcriptional activity of p53, down regulates caspase-3 activity through -crystallin (CRYAB), and directly antagonist to caspase-7 in glioblastoma (GBM). However, whether BH3-like domain of Bcl2L12 involved in the apoptosis regulation in GBM is still unknown. |
Specific aims: Therefore, in this study, we basically investigate whether Bcl2L12 harbors a functional BH3-like domain involved in its anti-apoptotic role in GBM through interacting with pro-survival proteins of Bcl2 family such as Bcl-xL and Bcl2. Besides, we tested whether this BH3-like domain can be as promising target for sensitizing drug response of GBM to temozolomide (TMZ).
Methods: To approach these aims, we firstly computational simulated to compare the similarity of secondary and 3D-structure of Bcl2L12 with others Bcl2 family proteins, especially focused on BH3-like domain. Second, site-directed mutagenesis and yeast two hybrid assay were performed to pinpoint the residues may critical for Bcl2L12-Bcl-xL, Bcl-xL-BH3 interaction. Third, to determine whether disrupt the interaction between Bcl2L12 BH3-like domain (192-240) and BH3 and/or hydrophobic groove of Bcl2 family members led to a reactivation of apoptotic events, GFP-tagged Bcl2L12 wt and Bcl2L12 BH3-mutant L213A and L217A were overexpressed in glioma cell lines under different treatments (TMZ and staurosporine). Lately, the BH3 mimetic agent, ABT-737, was implied to confirm BH3-like domain of Bcl2L12 do responsible for its anti-apoptotic role and TMZ-induced autophagy in glioma.
Results: Our results showed that Bcl2L12 share a highly similarity with Bcl2 family members at BH3 domain, which is a consensus core domain of “LXXXAE(D)” through bioinformatical analysis. Moreover, our data showed that the h1 (L213), h2 (L217) and h4 (I224) residues are essential for the Bcl2L12 interaction with Bcl-xL and Bcl2 in yeast two hybrid system. Ectopically overexpressed Bcl2L12 wt, h1 and h2 mutant in GBM cell lines resulted in lost repression of apoptotic markers (cleaved caspase-3, -9, cytochrome c and PARP) as well as downregulates autophagy markers (Beclin-1 and LC3-II) under either staurosporine (STS) or temozolomide (TMZ) treatment in U87MG cells.
Conclusion: Altogether, we established a model to demonstrate that Bcl2L12 L213 and L217 as critical residues within BH3-like domain that confers an anti-apoptotic role and may involve in TMZ-induced autophagy through interplaying with Bcl-xL and Bcl2 in glioma cells. We, here, reported for the first time that Bcl2L12 harbors BH3-like domain and as a target can be modulated to sensitize drug response of GBM to TMZ.