Nix Interacts with WIPI2 to Induce Mitophagy
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ABSTRACT:
SUBMITTER: Eric, Newman Bunker
PROVIDER: S-SCDT-10_15252-EMBJ_2023113491 | biostudies-other |
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Nix is a membrane-anchored outer mitochondrial protein that induces mitophagy. While Nix has an LC3-interacting (LIR) motif that binds to ATG8 proteins, it also contains a minimal essential region (MER) that induces mitophagy through an unknown mechanism. We used chemically induced dimerization (CID) to probe the mechanism of Nix-mediated mitophagy and found that both the LIR and MER are required for robust mitophagy. We find that the Nix MER interacts with the autophagy effector WIPI2 and recruits WIPI2 to mitochondria. The Nix LIR motif is also required for robust mitophagy and converts a homogeneous WIPI2 distribution on the surface of the mitochondria into puncta, even in the absence of ATG8s. Together, this work reveals unanticipated mechanisms in Nix-induced mitophagy and the elusive role of the MER, while also describing an interesting example of autophagy induction that acts downstream of the canonical initiation complexes.
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Project description:Determining what autophagy proteins bind to the MER domain of Nix
2023-06-30 | MSV000092324 | MassIVE
Project description:Mutations in the gene encoding Parkin are a major cause of recessive Parkinson's disease. Recent work has shown that Parkin translocates from the cytosol to depolarized mitochondria and induces their autophagic removal (mitophagy). However, the molecular mechanisms underlying Parkin-mediated mitophagy are poorly understood. Here, we investigated whether Parkin interacts with autophagy-regulating proteins. We purified Parkin and associated proteins from HEK293 cells using tandem affinity purification and identified the Parkin interactors using mass spectrometry. We identified the autophagy-promoting protein Ambra1 (activating molecule in Beclin1-regulated autophagy) as a Parkin interactor. Ambra1 activates autophagy in the CNS by stimulating the activity of the class III phosphatidylinositol 3-kinase (PI3K) complex that is essential for the formation of new phagophores. We found Ambra1, like Parkin, to be widely expressed in adult mouse brain, including midbrain dopaminergic neurons. Endogenous Parkin and Ambra1 coimmunoprecipitated from HEK293 cells, SH-SY5Y cells, and adult mouse brain. We found no evidence for ubiquitination of Ambra1 by Parkin. The interaction of endogenous Parkin and Ambra1 strongly increased during prolonged mitochondrial depolarization. Ambra1 was not required for Parkin translocation to depolarized mitochondria but was critically important for subsequent mitochondrial clearance. In particular, Ambra1 was recruited to perinuclear clusters of depolarized mitochondria and activated class III PI3K in their immediate vicinity. These data identify interaction of Parkin with Ambra1 as a key mechanism for induction of the final clearance step of Parkin-mediated mitophagy.
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Project description:Aims: Mitochondrial stress and dysfunction within the intestinal epithelium are known to contribute to the pathogenesis of inflammatory bowel disease (IBD). However, the importance of mitophagy during intestinal inflammation remains poorly understood. The primary aim of this study was to investigate how the mitophagy protein BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L/NIX) mitigates mitochondrial damage during intestinal inflammation in the hopes that these data will allow us to target mitochondrial health in the intestinal epithelium as an adjunct to immune-based treatment strategies. Results: In the intestinal epithelium of patients with ulcerative colitis, we found that NIX was upregulated and targeted to the mitochondria. We obtained similar findings in wild-type mice undergoing experimental colitis. An increase in NIX expression was found to depend on stabilization of hypoxia-inducible factor-1 alpha (HIF1α), which binds to the Nix promoter region. Using the reactive oxygen species (ROS) scavenger MitoTEMPO, we were able to attenuate disease and inhibit both HIF1α stabilization and subsequent NIX expression, suggesting that mitochondrially derived ROS are crucial to initiating the mitophagic response during intestinal inflammation. We subjected a global Nix-/- mouse to dextran sodium sulfate colitis and found that these mice developed worse disease. In addition, Nix-/- mice were found to exhibit increased mitochondrial mass, likely due to the inability to clear damaged or dysfunctional mitochondria. Innovation: These results demonstrate the importance of mitophagy within the intestinal epithelium during IBD pathogenesis. Conclusion: NIX-mediated mitophagy is required to maintain intestinal homeostasis during inflammation, highlighting the impact of mitochondrial damage on IBD progression.
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Project description:Elimination of dysfunctional mitochondria via mitophagy is essential for cell survival and neuronal functions. But, how impaired mitophagy participates in tissue-specific vulnerability in the brain remains unclear. Here, we find that striatal-enriched protein, Rhes, is a critical regulator of mitophagy and striatal vulnerability in brain. In vivo interactome and density fractionation reveal that Rhes coimmunoprecipitates and cosediments with mitochondrial and lysosomal proteins. Live-cell imaging of cultured striatal neuronal cell line shows Rhes surrounds globular mitochondria, recruits lysosomes, and ultimately degrades mitochondria. In the presence of 3-nitropropionic acid (3-NP), an inhibitor of succinate dehydrogenase, Rhes disrupts mitochondrial membrane potential (ΔΨ m ) and promotes excessive mitophagy and cell death. Ultrastructural analysis reveals that systemic injection of 3-NP in mice promotes globular mitochondria, accumulation of mitophagosomes, and striatal lesion only in the wild-type (WT), but not in the Rhes knockout (KO), striatum, suggesting that Rhes is critical for mitophagy and neuronal death in vivo. Mechanistically, Rhes requires Nix (BNIP3L), a known receptor of mitophagy, to disrupt ΔΨ m and promote mitophagy and cell death. Rhes interacts with Nix via SUMO E3-ligase domain, and Nix depletion totally abrogates Rhes-mediated mitophagy and cell death in the cultured striatal neuronal cell line. Finally, we find that Rhes, which travels from cell to cell via tunneling nanotube (TNT)-like cellular protrusions, interacts with dysfunctional mitochondria in the neighboring cell in a Nix-dependent manner. Collectively, Rhes is a major regulator of mitophagy via Nix, which may determine striatal vulnerability in the brain.
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Project description:Platelet activation requires fully functional mitochondria, which provide a vital energy source and control the life span of platelets. Previous reports have shown that both general autophagy and selective mitophagy are critical for platelet function. However, the underlying mechanisms remain incompletely understood. Here, we show that Nix, a previously characterized mitophagy receptor that plays a role in red blood cell maturation, also mediates mitophagy in platelets. Genetic ablation of Nix impairs mitochondrial quality, platelet activation, and FeCl3-induced carotid arterial thrombosis without affecting the expression of platelet glycoproteins (GPs) such as GPIb, GPVI, and αIIbβ3 Metabolic analysis revealed decreased mitochondrial membrane potential, enhanced mitochondrial reactive oxygen species level, diminished oxygen consumption rate, and compromised adenosine triphosphate production in Nix -/- platelets. Transplantation of wild-type (WT) bone marrow cells or transfusion of WT platelets into Nix-deficient mice rescued defects in platelet function and thrombosis, suggesting a platelet-autonomous role (acting on platelets, but not other cells) of Nix in platelet activation. Interestingly, loss of Nix increases the life span of platelets in vivo, likely through preventing autophagic degradation of the mitochondrial protein Bcl-xL. Collectively, our findings reveal a novel mechanistic link between Nix-mediated mitophagy, platelet life span, and platelet physiopathology. Our work suggests that targeting platelet mitophagy Nix might provide new antithrombotic strategies.
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Project description:To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation, and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4, an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.
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Project description:Activating KRAS mutations are found in nearly all cases of pancreatic ductal adenocarcinoma (PDAC), yet effective clinical targeting of oncogenic KRAS remains elusive. Understanding of KRAS-dependent PDAC-promoting pathways could lead to the identification of vulnerabilities and the development of new treatments. We show that oncogenic KRAS induces BNIP3L/NIX expression and a selective mitophagy program that restricts glucose flux to the mitochondria and enhances redox capacity. Loss of Nix restores functional mitochondria to cells, increasing demands for NADPH reducing power and decreasing proliferation in glucose-limited conditions. Nix deletion markedly delays progression of pancreatic cancer and improves survival in a murine (KPC) model of PDAC. Although conditional Nix ablation in vivo initially results in the accumulation of mitochondria, mitochondrial content eventually normalizes via increased mitochondrial clearance programs, and pancreatic intraepithelial neoplasia (PanIN) lesions progress to PDAC. We identify the KRAS-NIX mitophagy program as a novel driver of glycolysis, redox robustness, and disease progression in PDAC. SIGNIFICANCE: NIX-mediated mitophagy is a new oncogenic KRAS effector pathway that suppresses functional mitochondrial content to stimulate cell proliferation and augment redox homeostasis. This pathway promotes the progression of PanIN to PDAC and represents a new dependency in pancreatic cancer.This article is highlighted in the In This Issue feature, p. 1143.
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Project description:Mitophagy, the elimination of damaged mitochondria through autophagy, promotes neuronal survival in cerebral ischemia. Previous studies found deficient mitophagy in ischemic neurons, but the mechanisms are still largely unknown. We determined that BNIP3L/NIX, a mitophagy receptor, was degraded by proteasomes, which led to mitophagy deficiency in both ischemic neurons and brains. BNIP3L exists as a monomer and homodimer in mammalian cells, but the effects of homodimer and monomer on mitophagy are unclear. Site-specific mutations in the transmembrane domain of BNIP3L (S195A and G203A) only formed the BNIP3L monomer and failed to induce mitophagy. Moreover, overexpression of wild-type BNIP3L, in contrast to the monomeric BNIP3L, rescued the mitophagy deficiency and protected against cerebral ischemic injury. The macroautophagy/autophagy inhibitor 3-MA and the proteasome inhibitor MG132 were used in cerebral ischemic brains to identify how BNIP3L was reduced. We found that MG132 blocked the loss of BNIP3L and subsequently promoted mitophagy in ischemic brains. In addition, the dimeric form of BNIP3L was more prone to be degraded than its monomeric form. Carfilzomib, a drug for multiple myeloma therapy that inhibits proteasomes, reversed the BNIP3L degradation and restored mitophagy in ischemic brains. This treatment protected against either acute or chronic ischemic brain injury. Remarkably, these effects of carfilzomib were abolished in bnip3l-/- mice. Taken together, the present study linked BNIP3L degradation by proteasomes with mitophagy deficiency in cerebral ischemia. We propose carfilzomib as a novel therapy to rescue ischemic brain injury by preventing BNIP3L degradation.Abbreviations: 3-MA: 3-methyladenine; AAV: adeno-associated virus; ATG7: autophagy related 7; BCL2L13: BCL2-like 13 (apoptosis facilitator); BNIP3L/NIX: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CFZ: carfilzomib; COX4I1: cytochrome c oxidase subunit 4I1; CQ: chloroquine; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; I-R: ischemia-reperfusion; MAP1LC3A/LC3A: microtube-associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtube-associated protein 1 light chain 3 beta; O-R: oxygen and glucose deprivation-reperfusion; OGD: oxygen and glucose deprivation; PHB2: prohibitin 2; pMCAO: permanent middle cerebral artery occlusion; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; PT: photothrombosis; SQSTM1: sequestosome 1; tMCAO: transient middle cerebral artery occlusion; TOMM20: translocase of outer mitochondrial membrane 20; TTC: 2,3,5-triphenyltetrazolium hydrochloride.
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Project description:Stress-induced glucocorticoids disturb mitochondrial bioenergetics and dynamics; however, instead of being removed via mitophagy, the damaged mitochondria accumulate. Therefore, we investigate the role of glucocorticoids in mitophagy inhibition and subsequent synaptic defects in hippocampal neurons, SH-SY5Y cells, and ICR mice. First, we observe that glucocorticoids decrease both synaptic density and vesicle recycling due to suppressed mitophagy. Screening data reveal that glucocorticoids downregulate BNIP3-like (BNIP3L)/NIX, resulting in the reduced mitochondrial respiration function and synaptic density. Notably, we find that glucocorticoids direct the glucocorticoid receptor to bind directly to the PGC1α promoter, downregulating its expression and nuclear translocation. PGC1α downregulation selectively decreases NIX-dependent mitophagy. Consistent with these results, NIX enhancer pre-treatment of a corticosterone-exposed mouse elevates mitophagy and synaptic density in hippocampus, improving the outcome of a spatial memory task. In conclusion, glucocorticoids inhibit mitophagy via downregulating NIX and that NIX activation represents a potential target for restoring synapse function.
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