Faculty Opinions recommendation of A bacterial inhibitor of host programmed cell death defenses is an E3 ubiquitin ligase.

Autophagy plays an important role in cellular quality control and is responsible for removing protein aggregates and dysfunctional organelles. BNIP3 is an atypical BH3-only protein which is known to cause mitochondrial dysfunction and cell death in the myocardium. Interestingly, BNIP3 can also protect against cell death by promoting removal of dysfunctional mitochondria via autophagy (mitophagy). We have previously reported that BNIP3 is a potent inducer of mitophagy in cardiac myocytes and that BNIP3 contains an LC3 Interacting Region (LIR) that binds to LC3 on the autophagosome, tethering the mitochondrion to the autophagosome for engulfment. However, the molecular mechanism(s) underlying BNIP3-mediated mitophagy are still unclear. In this study, we discovered that BNIP3 can mediate mitochondrial clearance in cells even in the absence of a functional autophagy pathway. We found that overexpression of BNIP3 led to significant clearance of mitochondria in both wild type (WT) and autophagy deficient Atg5-/- MEFs. BNIP3 caused an increase in LC3II levels in WT MEFs, indicating increased formation of autophagosomes. In contrast, LC3II was undetectable in Atg5-/- MEFs. Furthermore, we found that BNIP3-mediated clearance in WT and Atg5-/- MEFs did not require the presence of Parkin, an E3 ubiquitin ligase which plays a critical role in clearing dysfunctional mitochondria in cells. Also, overexpression of Parkin did not enhance BNIP3-mediated mitochondrial clearance. When investigating activation of alternative cellular degradation pathways, we found that BNIP3 induced activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. Mutating the LC3 binding site in BNIP3 did not interfere with the activation of the endosomal pathway and clearance of mitochondria in Atg5-/- MEFs. Thus, these findings suggest that BNIP3 can promote clearance of mitochondria via multiple pathways in cells. The role of autophagy in removing mitochondria is already well established and we are currently exploring the roles of the endosomal and alternative autophagy pathways in BNIP3-mediated mitochondrial clearance in myocytes.

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Abstract 243: The E3 Ubiquitin Ligase Parkin Mediates Clearance of Damaged Mitochondria via Two Distinct Pathways

Circulation Research ◽

10.1161/res.117.suppl_1.243 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Babette C Hammerling ◽

Melissa Q Cortez ◽

Rita A Hanna ◽

Eileen R Gonzalez ◽

Åsa B Gustafsson

Keyword(s):

Cell Death ◽

Ubiquitin Ligase ◽

Ischemia Reperfusion ◽

E3 Ubiquitin Ligase ◽

Dominant Negative ◽

Mitochondrial Ros ◽

Late Endosomes ◽

Early Endosomes ◽

Important Regulator ◽

Rapid Activation

Damaged mitochondria release reactive oxygen species and pro-death factors which can lead to loss of cardiac myocytes. To protect against such damage, myocytes have developed several mechanisms of quality control that act both on the protein and organelle levels. We have previously identified the E3 ubiquitin ligase Parkin as an important regulator of mitochondrial clearance via autophagy in the myocardium. Here, we report that Parkin can also mediate clearance of mitochondria via the endosomal-lysosomal pathway. We found that Parkin promotes clearance of damaged mitochondria in both wild type (WT) and autophagy-deficient Atg5 knockout mouse embryonic fibroblasts (MEFs) treated with the mitochondria uncoupler FCCP. Mitochondrial damage leads to rapid activation of the endosomal-lysosomal pathway in both WT and Atg5-/- MEFs. We further observed increased activation of Rab5, a protein involved in early endosome formation, in both WT and Atg5-/- MEFs after treatment with FCCP. In addition, we observed sequestration of damaged mitochondria in Rab5+ and Rab7+ early and late endosomes, respectively. Mitochondria also colocalized with Lamp2+ vesicles in Atg5-/- MEFs indicating that the mitochondria are ultimately being delivered to the lysosomes for degradation. Overexpression of Rab5S34N, a dominant negative of Rab5, reduces FCCP-mediated clearance and increases cell death in Atg5-/- MEFs. Pharmacological inhibition of the endosomal-lysosomal pathway also results in increased FCCP-mediated cell death. Furthermore, we confirmed that FCCP treatment or simulated ischemia reperfusion exposure induces Rab5 activation with subsequent mitochondrial sequestration in early endosomes in neonatal myocytes. Interestingly, the activation of Rab5 is abrogated in the presence of the mitochondrial targeted antioxidant Mito-Tempo, suggesting that mitochondrial ROS is involved in the activation the endosomal pathway. Mitochondrial clearance via this pathway is also dependent on Parkin, as FCCP treatment fails to activate Rab5 and induce mitochondrial clearance in both WT and Atg5-/- MEFS in the absence of Parkin. Thus, our data suggest that Parkin can mediate clearance of damaged mitochondria via two distinct pathways in cells.

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