In this study, we revealed a novel molecular mechanism by which the live/death decision of human cells is regulated by the ASB11-mediated ubiquitination and proteasomal degradation of BIK. In this model, ASB11 was identified as the SRS that recruits BIK to the Cul5-based ubiquitin E3 ligase complex for BIK ubiquitination. This conclusion is based on several lines of evidence. First, the interaction of ASB11 and BIK was demonstrated by the Co-IP and in vitro binding assay. Second, in vitro ubiquitination assay demonstrated the capacity of reconstituted ElonginBC-Cul5-ROC2-ASB11 E3 complex to promote polyubiquitination of BIK in vitro. Third, overexpression of ASB11 reduced the stability and abundance of BIK protein in a proteasome dependent manner, indicating that ASB11 promoted BIK degradation through ubiquitin-proteasome system. Together, our study identified ASB11-based Cul5 complex as an ubiquitin ligase for BIK.
After the discovery of ASB11-mediated BIK ubiquitination/proteasomal degradation pathway, we further investigated the physiological roles and biochemical regulation of this machinery. In the DNA damage-induced cell death, it is known that BIK is transcriptionally upregulated by p53 to promote apoptosis. Here, we identified an additional mechanism that facilitates BIK accumulation upon DNA damage through the blockage of ASB11-mediated proteasomal degradation of BIK protein and indicated the contribution of this BIK stabilization to DNA damage-induced apoptosis. These conclusions are based on following findings. First, ASB11 transcription is repressed in response to several DNA damage agents through a p53-dependent manner. Second, DNA damage downregulated BIK ubiquitination level in a p53-dependent manner.
Third, DNA damage diminished BIK proteasomal degradation through a p53-dependent manner. Consistent with these observations, ASB11 overexpression inhibits DNA damage-induced apoptosis and this effect is dependent on the formation of ASB11-containing Cul5 complex. Despite these findings, the detailed mechanism by which ASB11 transcription is repressed by DNA damage/p53 pathway remains to be investigated. It is still unknown that whether ASB11 is directly targeted or indirectly regulated by p53. The analysis performed by PROMO, an online bioinformatics program specifically designed to identify putative transcription factor binding sites, indicated the existence of putative p53 binding sites on the promoter of ASB11.
However, genome-wide studies did show that p53 represses genes expression mainly through the indirect but not the direct pathways[127]. In the future, the putative p53 binding sites will be examined by employing the ChIP assay to further investigate the mechanism of p53-suppressed ASB11 expression.
In addition to the DNA damage response, we found that ER stress can also regulate the expression of ASB11 and BIK. However, in contrast to the downregulation of ASB11 in DNA damage response, ASB11 mRNA is upregulated by UPR in response to the ER stress. We demonstrated that ASB11 is transcriptionally upregulated through IRE1-XBP1 pathway in 293T cells treated with ER stress inducers tunicamycin and thapsigargin. Consistent with this result, the ubiquitination and proteasomal degradation of BIK were both induced by tunicamycin treatment through IRE1-XBP1 pathway.
These findings implied that ASB11 is involved in the adaptive response of UPR by mediating BIK ubiquitination and degradation. Based on the fact that BIK was reported to be binding and inhibited by the ER stress sensor GRP78 [68], it is interesting to know
stress response, right after GRP78 is occupied by the unfolded proteins. If so, ASB11 may play a critical role in the pro-survival pathway in the early state of UPR. However, more studies are necessary to support our hypothesis. In the future, we will exam the influence of ASB11 on the sensitivity of cells to ER stress agents. If ASB11 indeed plays critical role in the pro-survival adaptive response of UPR, knockdown of ASB11 should sensitize cells to the ER stress agents. Furthermore, we will evaluate whether the elevated ubiquitination and proteasomal degradation of BIK is dependent on the upregulation of ASB11 in the ER stress response. Finally, the mechanism by which XBP1 regulates ASB11 will also be determined. XBP1 is a transcription factor and PROMO analysis identified a number of XBP1 binding sites on ASB11 promoter.
Future studies will determine the critical region in this promoter responsible of XBP1-mediated transactivation and the direct or indirect role of XBP1 in promoting ASB11 transcription.
In this thesis, we uncover the regulatory mechanisms for ASB11-based Cul5 ubiquitin ligase in response to different stress conditions to alter the stability of a proapoptotic protein BIK. The opposite regulation of ASB11 by DNA damage and UPR would lead to opposite outcomes in the live/death decision of cells. Besides the understanding of basic molecular mechanism of cell fate decision mediated by ASB11, our study also offers important clinical implications by exploiting this function of ASB11. For instance, our finding of the pro-survival role of ASB11 in response to DNA damage agents suggests that ASB11 can be a novel target for combination therapy to sensitize cancer cells to chemotherapeutic agents. This notion is supported by a previous study demonstrating that treatment of proteasome inhibitor bortezomib induces BIK protein accumulation to sensitize otherwise resistant head and neck squamous cell
carcinomas to cisplatin treatment [128]. In contrast, inhibition of ASB11 may be a beneficial way to overcome the adaptive response of UPR and then sensitize cancer cell to ER stress inducers. Furthermore, UPR and ER stress-induced apoptosis are also be involved in pathologic conditions other than cancer, such as neurodegenerative diseases, diabetes mellitus, and ischemia. It is interesting to know that what is, if any, the role of ASB11-mediated BIK ubiquitination in those conditions [129].
In conclusion, our study uncovers a BIK ubiquitination and degradation mechanism mediated by ElonginBC-Cul5-ROC2-ASB11 ubiquitin ligase complex. We further discover the opposite regulations of ASB11 by DNA damage and ER stress to affect the proteostasis of BIK thereby influencing on the live/death decisions of cells under these stressed conditions (Fig. 16). Based on our results, we suggest that ASB11 can be a potential target for certain diseases, such as cancer and neurodegenerative diseases. Together, this thesis provides novel and interesting insights into the DNA damage-induced and ER stress-induced apoptosis pathways and implies new therapeutic strategies for devastating human diseases.