Drug manipulation of pro- and anti- apoptotic Bcl-2 proteins in the regulation of Parkin-mediated mitophagy in human breast cancer cells
Embargo until
2022-05-01
Date
2018-04-05
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Publisher
Johns Hopkins University
Abstract
Intrinsic apoptosis is a mode of programmed cell death with fundamental steps centralized to mitochondrial compartments. One of the most critical steps in apoptotic induction is the release of pro-death intermembrane space-localized proteins in a process termed mitochondrial outer membrane permeabilization, or MOMP. During MOMP, pro-apoptotic Bcl-2 family proteins Bax and Bak form pores in outer membranes of fragmented mitochondria. Mitochondrial fragmentation is also associated with deteriorated organelle function, suggesting that apoptosis signaling may crosstalk with mitochondrial quality control programs (i.e. mitophagy). Because cancer cells often manipulate apoptosis signaling to enable hyperproliferative growth, it is important to understand the extent of this potential crosstalk. Intriguingly, recent findings indicate interactions between apoptosis and mitophagy regulators. Parkin, an E3 ligase that can regulate mitophagy, translocates to depolarized mitochondria. Parkin ubiquitylates multiple proteins leading to the recruitment of autophagy receptors and initiation of mitophagy. Anti-apoptotic Bcl-2 family protein overexpression can block Parkin translocation, and in some cases, mitophagy. However, the exact mechanism, extent, and sufficiency of crosstalk between apoptosis and mitophagy pathways is unclear. In this study, we utilize direct and indirect apoptosis activating compounds to further dissect mitophagy-apoptosis pathway crosstalk. We found that direct and indirect activation of apoptosis induced by compounds Bam7 and Venetoclax enhanced mitophagy in Parkin overexpressing cells. These results are consistent with previous findings, which showed enhanced mitophagic capacity after BH3-only protein overexpression, and decreased mitophagic capacity after anti-apoptotic Bcl-2 protein overexpression. Utilization of additional pro-apoptotic compounds led to an interesting observation. While we show that administration of the recently discovered pro-apoptotic compound Raptinal correlates with hallmarks of apoptosis (e.g. Bax clustering, PARP cleavage, and cellular vacuolization), Raptinal treatment in Parkin mitophagy assays resulted in signs of decreased mitophagic capacity. Notably, we detected comparatively increased polyubiquitin levels in Raptinal-treated cells after challenge with mitochondrial uncouplers. Although further studies on the mechanism of apoptotic induction by Raptinal need to be completed, these findings suggest a divergence in the shared regulation of apoptosis and mitophagy. In conclusion, this work demonstrates the sufficiency of basal Bcl-2 family protein levels in facilitating crosstalk between apoptosis and Parkin-mediated mitophagy.
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Keywords
Mitophagy, Parkin, Apoptosis, Raptinal, Cell Death, Mitochondria