Nix Is Critical to Two Distinct Phases of Mitophagy, Reactive Oxygen Species-mediated Autophagy Induction and Parkin-Ubiquitin-p62-mediated Mitochondrial Priming
ABSTRACT Damaged mitochondria can be eliminated by autophagy, i.e. mitophagy, which is important for cellular homeostasis and cell survival. Despite the fact that a number of factors have been found to be important for mitophagy in mammalian cells, their individual roles in the process had not been clearly defined. Parkin is a ubiquitin-protein isopeptide ligase able to translocate to the mitochondria that are to be removed. We showed here in a chemical hypoxia model of mitophagy induced by an uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP) that Parkin translocation resulted in mitochondrial ubiquitination and p62 recruitment to the mitochondria. Small inhibitory RNA-mediated knockdown of p62 significantly diminished mitochondrial recognition by the autophagy machinery and the subsequent elimination. Thus Parkin, ubiquitin, and p62 function in preparing mitochondria for mitophagy, here referred to as mitochondrial priming. However, these molecules were not required for the induction of autophagy machinery. Neither Parkin nor p62 seemed to affect autophagy induction by CCCP. Instead, we found that Nix was required for the autophagy induction. Nix promoted CCCP-induced mitochondrial depolarization and reactive oxygen species generation, which inhibited mTOR signaling and activated autophagy. Nix also contributed to mitochondrial priming by controlling the mitochondrial translocation of Parkin, although reactive oxygen species generation was not involved in this step. Deletion of the C-terminal membrane targeting sequence but not mutations in the BH3 domain disabled Nix for these functions. Our work thus distinguished the molecular events responsible for the different phases of mitophagy and placed Nix upstream of the events.
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- "Downstream of the PINK1/Parkin Signaling Pathway LC3-containing autophagosomes can be efficiently recruited to mitochondria by P62 (Ding et al., 2010; Geisler et al., 2010; Huang et al., 2011). To investigate if PMI treatment corresponded with increased mitochondrial-associated P62, a similar confocal imaging experiment was performed (Figure 3A) to measure colocalization of P62 with the mitochondrial network. "
ABSTRACT: Mitophagy is central to mitochondrial and cellular homeostasis and operates via the PINK1/Parkin pathway targeting mitochondria devoid of membrane potential (DJ m) to autophagosomes. Although mi-tophagy is recognized as a fundamental cellular process, selective pharmacologic modulators of mitophagy are almost nonexistent. We developed a compound that increases the expression and signaling of the autophagic adaptor molecule P62/ SQSTM1 and forces mitochondria into autophagy. The compound, P62-mediated mitophagy inducer (PMI), activates mitophagy without recruiting Parkin or collapsing DJ m and retains activity in cells devoid of a fully functional PINK1/Parkin pathway. PMI drives mitochondria to a process of quality control without compromising the bio-energetic compe-tence of the whole network while exposing just those organelles to be recycled. Thus, PMI circumvents the toxicity and some of the nonspecific effects associ-ated with the abrupt dissipation of DJ m by iono-phores routinely used to induce mitophagy and represents a prototype pharmacological tool to investigate the molecular mechanisms of mitophagy. INTRODUCTIONChemistry & biology 11/2014; 21(11):1585–1596. · 6.65 Impact Factor
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- "(2013); Yamano and Youle (2013); Youle and Narendra (2011) PARKIN Mitochondrial membrane depolarizers (CCCP, valinomycin) Accumulation of misfolded proteins in the mitochondrial matrix Ubiquitination of mitochondrial proteins e.g. VDAC, Mfn1, Mfn2,… PINK1 Mfn2 BNIP3 NIX VDAC1 HKI Miro1 Chen and Dorn (2013); Ding et al. (2010); Geisler et al. (2010); Kim et al. (2008); Lee et al. (2011); Okatsu et al. (2012); Wang et al. (2011) TBC1D15 Mitochondrial depolarizers (valinomycin) Link autophagosomal isolation membrane to mitochondrial cargo size via inhibition of Rab7 LC3/ GABARAP Fis1 TBC1D17 Rab7 Onoue et al. (2013); Yamano et al. (2014) TBC1D17 Mitochondrial depolarizers (valinomycin) Link autophagosomal isolation membrane to mitochondrial cargo size via inhibition of Rab7 TBC1D15 Yamano et al. (2014) TOMM7 Mitochondrial membrane depolarizers (CCCP) Stabilization of PINK1 on the outer mitochondrial membrane PINK1 Hasson et al. (2013) HSPA1L Mitochondrial membrane depolarizers (CCCP) Positive regulator of PARKIN translocation to damaged mitochondria PARKIN Hasson et al. (2013) BAG4 Mitochondrial membrane depolarizers (CCCP) Negative regulator of PARKIN translocation to damaged mitochondria PARKIN Hasson et al. (2013) SIAH3 Mitochondrial membrane depolarizers (CCCP) Mitochondrial protein that inhibits PINK1 accumulation after mitochondrial insult Hasson et al. (2013) PARL Mitochondrial membrane depolarizers (CCCP) Mitochondrial protein that cleaves PINK1 under basal conditions PINK1 Jin and Youle (2013); Jin et al. (2010) NIX Mitochondrial membrane depolarizers (CCCP) Mitochondrial clearance during erythrocyte maturation Hypoxia Differentiation of red blood cells Increase depolarization and ROS production of mitochondria Induction of autophagic machinery Mitophagic receptor Recruitment of Rheb to mitochondria Recruitment of PARKIN to mitochondria LC3/ GABARAP PARKIN Ding et al. (2010); Lee et al. (2011); Mazure and Pouyssegur (2009); Novak et al. (2010); Sandoval et al. (2008); Schweers et al. (2007) BNIP3 Hypoxia-reoxygenation Mitochondrial membrane depolarizers (CCCP) Induction of autophagic machinery Recruitment of PARKIN to mitochondria Recruitment of DRP1 to mitochondria Induction of degradation of mitochondrial proteins Mitophagic receptor Mitophagic receptor LC3/ GABARAP Bcl2 Rheb PARKIN Hanna et al. (2012); Lee et al. (2011); Li et al. (2007); Mazure and Pouyssegur (2009); Zhu et al.(2013) FUNDC1 Hypoxia Mitochondrial membrane depolarizers (FCCP) LC3/ GABARAP ULK1 Liu et al. (2012); Wu et al. (2014) "
ABSTRACT: Autophagy, or self-eating, is the most extensively studied lysosomal degradation pathway for the recycling of obsolete or damaged cytoplasmic materials, including proteins and organelles. Although this pathway was initially thought to function as trafficking system for ‘in bulk’ degradation by the lysosomes of cytoplasmic material, it is now widely appreciated that cargo selection by the autophagic machinery is a major process underlying the cytoprotective or –possibly- pro-death functions ascribed to this catabolic process. Indeed increasing evidence suggests that in mammalian cells the removal of dysfunctional or aged mitochondria occurs through a selective degradation pathway known as ‘mitophagy’. Due to the crucial role of mitochondria in energy metabolism, redox control and cell survival/death decision, deregulated mitophagy can potentially impact a variety of crucial cell autonomous and non-autonomous processes. Accumulating evidence indicates that during malignant transformation aggressive cancers hijack autophagy to preserve energy fitness and to acquire the plasticity required to adapt to the hostile microenvironment. However, whether and how mitophagy contributes to carcinogenesis, which pathways regulates this process in the cancer cells and how cancer cell-mitophagy impacts and modifies the tumor microenvironment and therapeutic responses, remain largely unanswered issues. In this review, we discuss novel paradigms and pathways regulating mitophagy in mammalian cells and the impact this process might have on one of the most dreadful human malignancies, melanoma.Mitochondrion 11/2014; 19. DOI:10.1016/j.mito.2014.07.003 · 3.25 Impact Factor
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- "NAC can also prevent hypoxia-induced mitophagy, suggesting that ROS are involved (unpublished observations). Interestingly, it has been shown that ROS and the mitophagy receptor NIX/BNIP3L promote the induction and initiation of mitophagy by enhancing the translocation of Parkin onto damaged mitochondria77, suggesting that PINK1/Parkin-mediated mitophagy and the receptor-mediated pathways could be interdependent. "
ABSTRACT: Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease.Cell Research advance online publication 6 Jun 2014; doi:10.1038/cr.2014.75.Cell Research 06/2014; 24(7). DOI:10.1038/cr.2014.75 · 12.41 Impact Factor