Most cellular stress responses converge on the mitochondria. Consequently, the mitochondria must rapidly respond to maintain cellular homeostasis and physiological demands by fine-tuning a plethora ...of mitochondria-associated processes. The outer mitochondrial membrane (OMM) proteins are central to mediating mitochondrial dynamics, coupled with continuous fission and fusion. These OMM proteins also have vital roles in controlling mitochondrial quality and serving as mitophagic receptors for autophagosome enclosure during mitophagy. Mitochondrial fission segregates impaired mitochondria in smaller sizes from the mother mitochondria and may favor mitophagy for eliminating damaged mitochondria. Conversely, mitochondrial fusion mixes dysfunctional mitochondria with healthy ones to repair the damage by diluting the impaired components and consequently prevents mitochondrial clearance via mitophagy. Despite extensive research efforts into deciphering the interplay between fission-fusion and mitophagy, it is still not clear whether mitochondrial fission essentially precedes mitophagy. In this review, we summarize recent breakthroughs concerning OMM research, and dissect the functions of these proteins in mitophagy from their traditional roles in fission-fusion dynamics, in response to distinct context, at the intersection of the OMM platform. These insights into the OMM proteins in mechanistic researches would lead to new aspects of mitochondrial quality control and better understanding of mitochondrial homeostasis intimately tied to pathological impacts.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Mitophagy is the selective autophagic targeting and removal of dysfunctional mitochondria. While PINK1/Parkin-dependent mitophagy is well-characterized, PINK1/Parkin-independent route is poorly ...understood. Using structure illumination microscopy (SR-SIM), we demonstrate that the SNARE protein Syntaxin 17 (STX17) initiates mitophagy upon depletion of outer mitochondrial membrane protein Fis1. With proteomics analysis, we identify the STX17-Fis1 interaction, which controls the dynamic shuffling of STX17 between ER and mitochondria. Fis1 loss results in aberrant STX17 accumulation on mitochondria, which exposes the N terminus and promotes self-oligomerization to trigger mitophagy. Mitochondrial STX17 interacts with ATG14 and recruits core autophagy proteins to form mitophagosome, followed by Rab7-dependent mitophagosome-lysosome fusion. Furthermore, Fis1 loss impairs mitochondrial respiration and potentially sensitizes cells to mitochondrial clearance, which is mediated through canonical autophagy machinery, closely linking non-selective macroautophagy to mitochondrial turnover. Our findings uncover a PINK1/Parkin-independent mitophagic mechanism in which outer mitochondrial membrane protein Fis1 regulates mitochondrial quality control.
Mitochondrial DNA (mtDNA) escaping stressed mitochondria provokes inflammation via cGAS-STING pathway activation and, when oxidized (Ox-mtDNA), it binds cytosolic NLRP3, thereby triggering ...inflammasome activation. However, it is unknown how and in which form Ox-mtDNA exits stressed mitochondria in non-apoptotic macrophages. We found that diverse NLRP3 inflammasome activators rapidly stimulated uniporter-mediated calcium uptake to open mitochondrial permeability transition pores (mPTP) and trigger VDAC oligomerization. This occurred independently of mtDNA or reactive oxygen species, which induce Ox-mtDNA generation. Within mitochondria, Ox-mtDNA was either repaired by DNA glycosylase OGG1 or cleaved by the endonuclease FEN1 to 500–650 bp fragments that exited mitochondria via mPTP- and VDAC-dependent channels to initiate cytosolic NLRP3 inflammasome activation. Ox-mtDNA fragments also activated cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA. Understanding this process will advance the development of potential treatments for chronic inflammatory diseases, exemplified by FEN1 inhibitors that suppressed interleukin-1β (IL-1β) production and mtDNA release in mice.
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•Ca2+ uptake via MCU triggers IMM mPTP opening to induce OMM VDAC oligomerization•Ox-mtDNA is repaired by OGG1 or cleaved by FEN1 to fragments that exit mitochondria•Cytosolic Ox-mtDNA activates NLRP3 inflammasome and cGAS-STING and escapes cells•mtOGG1 and FEN1 inhibitors suppress acute peritonitis and reduce circulating mtDNA
Ox-mtDNA enables NLRP3 inflammasome activation, but how Ox-mtDNA reaches the cytoplasm is unclear. In this issue of Immunity, Xian et al. show that Ox-mtDNA cleaved by FEN1 escapes mitochondria via mPTP and VDAC channels to activate NLRP3 inflammasome and STING.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mitochondrial dynamics is highly implicated in a plethora of cellular processes including apoptosis and mitophagy. However, little is known about the scope and precise functions of mitochondrial ...dynamics proteins for mitochondrial quality control and cellular homeostasis. Whether mitochondrial dynamics proteins serve in cellular processes reliant on mitochondrial fission-fusion is still not fully explored. MIEF1/MiD51 (mitochondrial elongation factor 1) is known to promote mitochondrial fission via the recruitment of GTPase protein DNM1L/DRP1 (dynamin 1 like), but the fundamental understandings of MIEF1 for mitochondrial-dependent cellular processes are largely elusive. Here, we report novel roles of MIEF1 in responding to apoptotic stimuli and mitochondrial damage. Given our result that staurosporine (STS) treatment induced the degradation of MIEF1 via the ubiquitin-proteasome system (UPS), we are motivated to explore the role of MIEF1 in apoptosis. MIEF1 loss triggered the imbalance of BCL2 family members on the mitochondria, consequently initiating the translocation of BAX onto the mitochondria, catalyzing the decrease of mitochondrial membrane potential and promoting the release of DIABLO/SMAC (diablo IAP-binding mitochondrial protein) and CYCS (cytochrome c, somatic). We further demonstrate that MIEF1 deficiency impaired mitochondrial respiration and induced mitochondrial oxidative stress, sensitizing cells to PINK1-PRKN-mediated mitophagy. The recruitment of PRKN to depolarized mitochondria modulated the UPS-dependent degradation of MFN2 (mitofusin 2) and FIS1 (fission, mitochondrial 1) specifically, to further promote mitophagy. Our findings uncover a bridging role of MIEF1 integrating cell death and mitophagy, unlikely dependent on mitochondrial dynamics, implying new insights to mechanisms determining cellular fate.
Abbreviations: ActD: actinomycin D; BAX: BCL2 associated X, apoptosis regulator; BAK1: BCL2 antagonist/killer 1; BCL2L1: BCL2 like 1; BMH: 1,6-bismaleimidohexane; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CHX: cycloheximide; CQ: chloroquine; CYCS: cytochrome c, somatic; DIABLO: diablo IAP-binding mitochondrial protein; DKO: double knockout; DNM1L/DRP1: dynamin 1 like; FIS1: fission, mitochondrial 1; GFP: green fluorescent protein; IP: immunoprecipitation; MFN1: mitofusin 1; MFN2: mitofusin 2; MG132: carbobenzoxy-Leu-Leu-leucinal; MIEF1/MiD51: mitochondrial elongation factor 1; MIEF2/MiD49: mitochondrial elongation factor 2; MOMP: mitochondrial outer membrane permeabilization; MTR: MitoTracker Red; OA: oligomycin plus antimycin A; OCR: oxygen consumption rate; OMM: outer mitochondrial membrane; PARP: poly(ADP-ribose) polymerase; PI: propidium iodide; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; SD: standard deviation; STS: staurosporine; TNF: tumor necrosis factor; UPS: ubiquitin-proteasome system; VDAC1: voltage dependent anion channel 1.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Acute respiratory distress syndrome (ARDS), an inflammatory condition with high mortality rates, is common in severe COVID-19, whose risk is reduced by metformin rather than other anti-diabetic ...medications. Given evidence of inflammasome assembly in post-mortem COVID-19 lungs, we asked whether and how metformin inhibits inflammasome activation and exerts its anti-inflammatory effect. We show that metformin inhibited NLRP3 inflammasome activation and interleukin (IL)-1β production in cultured and alveolar macrophages along with inflammasome-independent IL-6 secretion, thus attenuating lipopolysaccharide (LPS)- and SARS-CoV-2-induced ARDS. Metformin blocked LPS-induced ATP-dependent synthesis of the NLRP3 ligand mtDNA independently of AMP-activated protein kinase (AMPK) or NF-κB. Myeloid-specific ablation of LPS-induced cytidine monophosphate kinase 2 (CMPK2), which is rate limiting for mtDNA synthesis, reduced ARDS severity without a direct effect on IL-6. Thus, inhibition of ATP and mtDNA synthesis is sufficient for ARDS amelioration.
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•Metformin inhibits macrophage IL-1β and IL-6 production and blunts ARDS severity•Metformin inhibits cytosolic Ox-mtDNA production and NLRP3 inflammasome activation•By targeting ETCCI, metformin blocks macrophage ATP-dependent mtDNA synthesis•Myeloid targeted inhibition of mtDNA synthesis blunts IL-1β production and ARDS
The exact mechanism by which metformin exerts its anti-inflammatory effects is still not known. Xian et al. shows that metformin’s inhibition of ETCCI blocks ATP-dependent mtDNA synthesis, cytoplasmic ox-mtDNA generation and NLRP3 inflammasome activation in macrophages, independent of AMPK and NF-κB. By limiting IL-1β production, metformin blunts pulmonary inflammation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Prokaryotic in origin, mitochondrial DNA (mtDNA) housed in eukaryotic cells exists within an oxidative environment and is subject to inefficient damage repair.Present at high copy number, mtDNA is an ...abundant source of stress-induced alarmins.mtDNA released from stressed mammalian mitochondria is likely to be oxidized and cleaved by flap structure-specific endonuclease 1 (FEN1).Oxidized (Ox)-mtDNA release depends on the opening of mitochondrial pores.Liberated Ox-mtDNA fragments engage different pattern recognition receptors depending on their location (cytoplasm, endosome, or extracellular space) to trigger proinflammatory and type I interferon (IFN) responses that are either beneficial or can ignite human autoimmunity and chronic inflammation.
The immunoregulatory functions of mitochondria – key players in innate immunity – can depend on the generation of oxidized mitochondrial DNA (Ox-mtDNA) which escapes to the cytosol and activates the NLRP3 inflammasome and cGAS–STING signaling. Ox-mtDNA also enters the circulation, thus amplifying the initial inflammation. Although the sterile inflammatory response activated by Ox-mtDNA is beneficial in nature, sustained circulating Ox-mtDNA is detected in diverse auto-immunopathologies, potentially acting as a pathogenic driver.
Despite the emergence of mitochondria as key regulators of innate immunity, the mechanisms underlying the generation and release of immunostimulatory alarmins by stressed mitochondria remains nebulous. We propose that the major mitochondrial alarmin in myeloid cells is oxidized mitochondrial DNA (Ox-mtDNA). Fragmented Ox-mtDNA enters the cytosol where it activates the NLRP3 inflammasome and generates IL-1β, IL-18, and cGAS–STING to induce type I interferons and interferon-stimulated genes. Inflammasome activation further enables the circulatory release of Ox-mtDNA by opening gasdermin D pores. We summarize new data showing that, in addition to being an autoimmune disease biomarker, Ox-mtDNA converts beneficial transient inflammation into long-lasting immunopathology. We discuss how Ox-mtDNA induces short- and long-term immune activation, and highlight its homeostatic and immunopathogenic functions.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mitochondrial morphologies change over time and are tightly regulated by dynamic machinery proteins such as dynamin-related protein 1 (Drp1), mitofusion 1/2, and optic atrophy 1 (OPA1). However, the ...detailed mechanisms of how these molecules cooperate to mediate fission and fusion remain elusive. DAP3 is a mitochondrial ribosomal protein that involves in apoptosis, but its biological function has not been well characterized. Here, we demonstrate that DAP3 specifically localizes in the mitochondrial matrix. Knockdown of DAP3 in mitochondria leads to defects in mitochondrial-encoded protein synthesis and abnormal mitochondrial dynamics. Moreover, depletion of DAP3 dramatically decreases the phosphorylation of Drp1 at Ser-637 on mitochondria, enhancing the retention time of Drp1 puncta on mitochondria during the fission process. Furthermore, autophagy is inhibited in the DAP3-depleted cells, which sensitizes cells to different types of death stimuli. Together, our results suggest that DAP3 plays important roles in mitochondrial function and dynamics, providing new insights into the mechanism of a mitochondrial ribosomal protein function in cell death.
Background: Mitochondrial dynamics is important for regulating cellular physiological function.
Results: Depletion of DAP3 impairs mitochondrial-encoded protein synthesis and leads to mitochondrial fission.
Conclusion: DAP3 is essential for maintaining mitochondrial function.
Significance: DAP3 is the first mitochondrial ribosomal protein to be characterized that regulates mitochondrial dynamics.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Defective mitophagy linked to dysfunction in the proteins Parkin and PTEN-induced putative kinase 1 (PINK1) is implicated in the pathogenesis of Parkinson's disease. Although the mechanism by which ...Parkin mediates mitophagy in a PINK1-dependent manner is becoming clearer, the triggers for this mitophagy pathway remain elusive. Reactive oxygen species (ROS) have been suggested as such triggers, but this proposal remains controversial because ROS scavengers fail to retard mitophagy. Here we demonstrate that the role of ROS in mitophagy has been underappreciated as a result of the inefficiency of ROS scavengers to control ROS bursts after high-dose treatment with carbonyl cyanide m-chlorophenylhydrazone. Supporting this, combinatorial treatment with N-acetyl-l-cysteine and catalase substantially inhibited the ROS upsurge and PINK1-dependent Parkin translocation to mitochondria in response to carbonyl cyanide m-chlorophenylhydrazone treatment. In addition to the chemical mitophagy inducer, overexpression of voltage-dependent anion channel 1 (VDAC1) induced Parkin translocation to mitochondria, presumably by stimulating ROS generation. Similarly, combined N-acetyl-l-cysteine and catalase treatment also suppressed VDAC1-induced redistribution of Parkin. Alongside these observations, we also found that the elevated protein level of PINK1 was not necessary for Parkin translocation to mitochondria. Thus, our data suggest that ROS may act as a trigger for the induction of Parkin/PINK1-dependent mitophagy. In addition, our study casts doubt on the importance of protein quantity of PINK1 in the recruitment of Parkin to mitochondria.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Mitochondrial quality control is essential for cellular homeostasis and accumulating evidence show that mitochondria can be selectively targeted for autophagic degradation (mitophagy). Mitophagy ...allows for the removal of dysfunctional mitochondria, which is highly implicated in energetically demanding cells including muscle and nerve cells. However, despite the well characterization of PINK1/Parkin route of mitophagy, mechanisms concerning PINK1/Parkin‐independent mitophagy are still poorly understood. With imaging approaches including structure‐illumination microscopy (SR‐SIM), here we demonstrate that the autophagy protein Syntaxin 17 (STX17), initiates mitophagy upon the depletion of outer mitochondrial membrane protein Fis1. Using mass spectrometry analysis, we identify STX17 interacts with Fis1, which preferentially gatekeeps the dynamic shuffling of STX17 between ER and mitochondria. Loss of Fis1 results in the accumulation of STX17 on mitochondria and mitochondria associated membranes (MAM), exposing its N‐terminus to assemble and self‐oligomerize for mitophagy. Mitochondrial STX17 interacts with ATG14 and further recruits core autophagy proteins hierarchically to form mitophagosomes, followed by Rab7‐dependent mitophagosome‐lysosome fusion. In addition, our results reveal that Fis1 loss impairs mitochondrial metabolic function, and potentially sensitizes mitochondria to STX17‐mediated mitochondrial engulfment within autophagosomes, which is directly initiated through canonical autophagy machinery, closely linking non‐selective macroautophagy and mitochondria. Our findings uncover a novel PINK1/Parkin‐independent mitophagy mechanism, in which outer mitochondrial membrane protein Fis1 gates the elimination of mitochondria.
Support or Funding Information
This work is financially supported by grants Tier 2 MOE and NUS, Singapore to Y.‐C.L.
This is from the Experimental Biology 2019 Meeting. There is no full text article associated with this published in The FASEB Journal.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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