Cardiac myocytes undergo programmed cell death as a result of ischemia/reperfusion (I/R). One feature of I/R injury is the increased presence of autophagosomes. However, to date it is not known whether macroautophagy functions as a protective pathway, contributes to programmed cell death, or is an irrelevant event during cardiac I/R injury. We employed simulated I/R of cardiac HL-1 cells as an in vitro model of I/R injury to the heart. To assess macroautophagy, we quantified autophagosome generation and degradation (autophagic flux), as determined by steady-state levels of autophagosomes in relation to lysosomal inhibitor-mediated accumulation of autophagosomes. We found that I/R impaired both formation and downstream lysosomal degradation of autophagosomes. Overexpression of Beclin1 enhanced autophagic flux following I/R and significantly reduced activation of pro-apoptotic Bax, whereas RNA interference knockdown of Beclin1 increased Bax activation. Bcl-2 and Bcl-x_L were protective against I/R injury, and expression of a Beclin1 Bcl-2/-x_L binding domain mutant resulted in decreased autophagic flux and did not protect against I/R injury. Overexpression of Atg5, a component of the autophagosomal machinery downstream of Beclin1, did not affect cellular injury, whereas expression of a dominant negative mutant of Atg5 increased cellular injury. These results demonstrate that autophagic flux is impaired at the level of both induction and degradation and that enhancing autophagy constitutes a powerful and previously uncharacterized protective mechanism against I/R injury to the heart cell.

Oncogenic KRas reprograms pancreatic ductal adenocarcinoma (PDAC) cells to states which are highly resistant to apoptosis. Thus, a major preclinical goal is to identify effective strategies for killing PDAC cells. Artesunate (ART) is an anti-malarial that specifically induces programmed cell death in different cancer cell types, in a manner initiated by reactive oxygen species (ROS)-generation. In this study we demonstrate that ART specifically induced ROS- and lysosomal iron-dependent cell death in PDAC cell lines. Highest cytotoxicity was obtained in PDAC cell lines with constitutively-active KRas, and ART did not affect non-neoplastic human pancreatic ductal epithelial (HPDE) cells. We determined that ART did not induce apoptosis or necroptosis. Instead, ART induced ferroptosis, a recently described mode of ROS- and iron-dependent programmed necrosis which can be activated in Ras-transformed cells. Co-treatment with the ferroptosis inhibitor ferrostatin-1 blocked ART-induced lipid peroxidation and cell death, and increased long-term cell survival and proliferation. Importantly, analysis of PDAC patient mRNA expression indicates a dependency on antioxidant homeostasis and increased sensitivity to free intracellular iron, both of which correlate with Ras-driven sensitivity to ferroptosis. Overall, our findings suggest that ART activation of ferroptosis is an effective, novel pathway for killing PDAC cells.

BH3-only proteins integrate apoptosis and autophagy pathways, yet regulation and functional consequences of pathway cross-talk are not fully resolved. The BH3-only protein Bnip3 is an autophagy receptor that signals autophagic degradation of mitochondria (mitophagy) via interaction of its LC3-interacting region (LIR) with Atg8 proteins. Here we report that phosphorylation of serine residues 17 and 24 flanking the Bnip3 LIR promotes binding to specific Atg8 members LC3B and GATE-16. Using quantitative multispectral image-based flow cytometry, we demonstrate that enhancing Bnip3-Atg8 interactions via phosphorylation-mimicked LIR mutations increased mitochondrial sequestration, lysosomal delivery, and degradation. Importantly, mitochondria were targeted by mitophagy prior to cytochrome c release, resulting in reduced cellular cytochrome c release capacity. Intriguingly, pro-survival Bcl-x_L positively regulated Bnip3 binding to LC3B, sequestration, and mitochondrial autophagy, further supporting an anti-apoptotic role for Bnip3-induced mitophagy. The ensemble of these results demonstrates that the phosphorylation state of the Bnip3 LIR signals either the induction of apoptosis or pro-survival mitophagy.

Mitochondria are an essential source of ATP for cellular function, but when damaged, mitochondria generate a plethora of stress signals, which lead to cellular dysfunction and eventually programmed cell death. Thus, a major component of maintaining cellular homeostasis is the recognition and removal of dysfunctional mitochondria through autophagy-mediated degradation, i.e., mitophagy. Mitophagy further constitutes a developmental program, and undergoes a high degree of crosstalk with apoptosis. Reduced mitochondrial quality control is linked to disease pathogenesis, suggesting the importance of process elucidation as a clinical target. Recent work has revealed multiple mitophagy programs that operate independently or undergo crosstalk, and require modulated autophagy receptor activities at outer membranes of mitochondria. Here, we review these mitophagy programs, focusing on pathway mechanisms which recognize and target mitochondria for sequestration by autophagosomes, as well as mechanisms controlling pathway activities. Furthermore, we provide an introduction to the currently available methods for detecting mitophagy.

The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.