Cytoplasmic upregulation of Cyto c and AIF serve as biomarkers of mechanical asphyxia death.
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ABSTRACT:
SUBMITTER: Zhang H
PROVIDER: S-EPMC6684934 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The mechanosensitive channel MscS functions as an osmolyte emergency release-valve in the event of a sudden decrease in external environmental osmolarity. MscS has served as a paradigm for studying how channel proteins detect and respond to mechanical stimuli. However, the inter-domain interactions and structural rearrangements that occur in the MscS gating process remain largely unknown. Here, we determined the interactions between the transmembrane domain and cytoplasmic domain of MscS. Using in vivo cellular viability, single-channel electrophysiological recording, and cysteine disulfide trapping, we demonstrated that N117 of the TM3b helix and N167 of the Cyto-helix are critical residues that function at the TM3b-Cyto helix interface. In vivo downshock assays showed that double cysteine substitution at N117 and N167 failed to rescue the osmotic-lysis phenotype of cells in acute osmotic downshock. Single-channel recordings demonstrated that cysteine cross-linking of N117C and N167C led to a non-conductive channel. Consistently, coordination of the histidines of N117H and N167H caused a decrease in channel gating. Moreover, cross-linked N117 and N167 altered the gating of the severe gain-of-function mutant L109S. Our results demonstrate that N117-N167 interactions stabilize the inactivation state by an association of TM3b segments with β-domains of the cytoplasmic region, providing further insights into the gating mechanism of the MscS channel.
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Project description:Apoptosis-inducing factor (AIF) is a phylogenetically conserved redox-active flavoprotein that contributes to cell death and oxidative phosphorylation in Saccharomyces cerevisiae, Caenorhabditis elegans, mouse and humans. AIF has been characterized as a caspase-independent death effector that is activated by its translocation from mitochondria to the cytosol and nucleus. Here, we report the molecular characterization of AIF in Drosophila melanogaster, a species in which most cell deaths occur in a caspase-dependent manner. Interestingly, knockout of zygotic D. melanogaster AIF (DmAIF) expression using gene targeting resulted in decreased embryonic cell death and the persistence of differentiated neuronal cells at late embryonic stages. Although knockout embryos hatch, they undergo growth arrest at early larval stages, accompanied by mitochondrial respiratory dysfunction. Transgenic expression of DmAIF misdirected to the extramitochondrial compartment (DeltaN-DmAIF), but not wild-type DmAIF, triggered ectopic caspase activation and cell death. DeltaN-DmAIF-induced death was not blocked by removal of caspase activator Dark or transgenic expression of baculoviral caspase inhibitor p35, but was partially inhibited by Diap1 overexpression. Knockdown studies revealed that DeltaN-DmAIF interacts genetically with the redox protein thioredoxin-2. In conclusion, we show that Drosophila AIF is a mitochondrial effector of cell death that plays roles in developmentally regulated cell death and normal mitochondrial function.
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Project description:Aortic dissection and aneurysm are associated with abnormal hemodynamic loads originating from hypertension. Our previous study demonstrated that cyclic mechanical stretch (CMS, mimicked hypertension) caused the death of rat aortic smooth muscle cells (RASMCs) in a mitogen activated-protein kinases (MAPKs)-dependent manner. The current study investigated the effects of inducible nitric oxide synthase (iNOS) on CMS-induced RASMC death. cDNA microarrays for CMS-treated RASMCs showed that iNOS expression levels were increased in response to CMS. Real-time polymerase chain reaction (PCR) analysis demonstrated that this increase was p38 MAPK (p38)-dependent. NO production was also increased. This increase could be inhibited by p38 and iNOS inhibitors. Thus, CMS-induced iNOS synthesized NO. CMS-induced cell death in RASMCs was increased by the iNOS inhibitor but abrogated by the long-acting NO donor DETA-NONOate. Increased iNOS expression was confirmed in the abdominal aortic constriction mouse model. Signal transducers and activators of transcription 1 (STAT1) was activated in stretched RASMCs, and iNOS expression and NO production were inhibited by the STAT1 inhibitor nifuroxazide. Our findings suggest that RASMCs were protected by iNOS from CMS-stimulated cell death through the STAT1 and p38 signal pathways independently.
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Project description:Mitochondrial dysfunction and release of pro-apoptotic factors such as cytochrome c or apoptosis-inducing factor (AIF) from mitochondria are key features of neuronal cell death. The precise mechanisms of how these proteins are released from mitochondria and their particular role in neuronal cell death signaling are however largely unknown. Here, we demonstrate by fluorescence video microscopy that 8-10 h after induction of glutamate toxicity, AIF rapidly translocates from mitochondria to the nucleus and induces nuclear fragmentation and cell death within only a few minutes. This markedly fast translocation of AIF to the nucleus is preceded by increasing translocation of the pro-apoptotic bcl-2 family member Bid (BH3-interacting domain death agonist) to mitochondria, perinuclear accumulation of Bid-loaded mitochondria, and loss of mitochondrial membrane integrity. A small molecule Bid inhibitor preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated glutamate-induced neuronal cell death, as shown by experiments using Bid small interfering RNA (siRNA). Cell death induced by truncated Bid was inhibited by AIF siRNA, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that although caspase-3 was activated, specific caspase-3 inhibition did not protect neuronal cells against glutamate toxicity. In conclusion, Bid-mediated mitochondrial release of AIF followed by rapid nuclear translocation is a major mechanism of glutamate-induced neuronal death.
| S-EPMC2862690 | biostudies-literature
Project description:Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2 mut/mut), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca2+) overload in Dsg2 mut/mut hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2 mut/mut mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca2+ overload-induced necrosis. When cardiomyocytes differentiated from Dsg2 mut/mut embryonic stem cells (ES-CMs) were challenged with β-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2 mut/mut ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.
| S-EPMC8936193 | biostudies-literature
Project description:Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear protein that is activated by binding to DNA lesions and catalyzes poly(ADP-ribosyl)ation of nuclear acceptor proteins, including PARP1 itself, to recruit DNA repair machinery to DNA lesions. When excessive DNA damage occurs, poly(ADP-ribose) (PAR) produced by PARP1 is translocated to the cytoplasm, changing the activity and localization of cytoplasmic proteins, e.g., apoptosis-inducing factor (AIF), hexokinase, and resulting in cell death. This cascade, termed parthanatos, is a caspase-independent programmed cell death distinct from necrosis and apoptosis. In contrast, PARP1 is a substrate of activated caspases 3 and 7 in caspase-dependent apoptosis. Once cleaved, PARP1 loses its activity, thereby suppressing DNA repair. Caspase cleavage of PARP1 occurs within a nuclear localization signal near the DNA-binding domain, resulting in the formation of 24-kDa and 89-kDa fragments. In the present study, we found that caspase activation by staurosporine- and actinomycin D-induced PARP1 autopoly(ADP-ribosyl)ation and fragmentation, generating poly(ADP-ribosyl)ated 89-kDa and 24-kDa PARP1 fragments. The 89-kDa PARP1 fragments with covalently attached PAR polymers were translocated to the cytoplasm, whereas 24-kDa fragments remained associated with DNA lesions. In the cytoplasm, AIF binding to PAR attached to the 89-kDa PARP1 fragment facilitated its translocation to the nucleus. Thus, the 89-kDa PARP1 fragment is a PAR carrier to the cytoplasm, inducing AIF release from mitochondria. Elucidation of the caspase-mediated interaction between apoptosis and parthanatos pathways extend the current knowledge on mechanisms underlying programmed cell death and may lead to new therapeutic targets.
| S-EPMC7948984 | biostudies-literature
Project description:AimsThe aims of this study were to: (a) identify differences in serum and cerebrospinal fluid (CSF) glucocorticoids among episodic migraine (EM) and chronic migraine (CM) patients compared with controls; (b) determine longitudinal changes in serum glucocorticoids in CM patients; and (c) determine migraine-related clinical features contributing to glucocorticoid levels.MethodsSerum and CSF levels of cortisol and corticosterone were measured using liquid chromatography-mass spectrometry among adult patients with EM, CM, and controls. Serum and CSF samples were collected from 26 and four participants in each group, respectively. Serum glucocorticoids were measured at a second timepoint after 2 years among 10 of the CM patients, six of whom reverted to EM while four persisted as CM. Receiver operating characteristic (ROC) analysis was made to assess the migraine diagnostic performance of glucocorticoids. Regression analysis was conducted to determine the link between glucocorticoid levels and migraine-related clinical variables.ResultsCM patients exhibited significantly elevated serum and CSF levels of cortisol and corticosterone compared with controls and EM patients (age, sex, body mass index adjusted; Kruskal-Wallis p < 0.05). ROC showed area-under-curve of 0.89 to differentiate CM from EM. CM patients with remission had their serum glucocorticoids return to control or near EM levels (p < 0.05). Persistent CM showed unremitting serum glucocorticoids. Migraine frequency and disability contributed to increased cortisol, while pain self-efficacy predicted lower cortisol levels (p < 0.005).ConclusionEndogenous glucocorticoids may be biomarkers for migraine progression and for monitoring treatment response. Improving pain self-efficacy skills may help optimize endogenous glucocorticoid levels, which in turn may prevent migraine attacks.
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Project description:Delayed neuronal cell death largely contributes to the progressive infarct development and associated functional impairments after cerebral ischemia or brain trauma. Previous studies exposed a key role for the interaction of the mitochondrial protein apoptosis-inducing factor (AIF) and cytosolic cyclophilin A (CypA) in pathways of programmed cell death in neurons in vitro and in vivo. These studies suggested that pro-apoptotic activities of AIF, such as its translocation to the nucleus and subsequent DNA degradation, depend on the physical interaction of AIF with CypA. Hence, this protein complex may represent a new pharmacological target for inhibiting the lethal action of AIF on the brain tissue. In this study, we show that the AIF amino-acid residues 370-394 mediate the protein complex formation of AIF with CypA. The synthetic AIF(370-394) peptide inhibited AIF/CypA complex formation in vitro by binding CypA with a K(D) of 12 ?M. Further, the peptide exerted pronounced neuroprotective effects in a model of glutamate-induced oxidative stress in cultured HT-22 cells. In this model system of AIF-dependent cell death, the AIF(370-394) peptide preserved mitochondrial integrity, as detected by measurements of the mitochondrial membrane potential and quantification of mitochondrial fragmentation. Further, the AIF(370-394) peptide inhibited perinuclear accumulation of fragmented mitochondria, mitochondrial release of AIF to the nucleus and glutamate-induced cell death to a similar extent as CypA-siRNA. These data indicate that the targeting of the AIF-CypA axis is an effective strategy of neuroprotection.
| S-EPMC4040673 | biostudies-literature
Project description:BackgroundProgrammed nuclear death (PND), which is also referred to as nuclear apoptosis, is a remarkable process that occurs in ciliates during sexual reproduction (conjugation). In Tetrahymena thermophila, when the new macronucleus differentiates, the parental macronucleus is selectively eliminated from the cytoplasm of the progeny, concomitant with apoptotic nuclear events. However, the molecular mechanisms underlying these events are not well understood. The parental macronucleus is engulfed by a large autophagosome, which contains numerous mitochondria that have lost their membrane potential. In animals, mitochondrial depolarization precedes apoptotic cell death, which involves DNA fragmentation and subsequent nuclear degradation.ResultsWe focused on the role of mitochondrial apoptosis-inducing factor (AIF) during PND in Tetrahymena. The disruption of AIF delays the normal progression of PND, specifically, nuclear condensation and kilobase-size DNA fragmentation. AIF is localized in Tetrahymena mitochondria and is released into the macronucleus prior to nuclear condensation. In addition, AIF associates and co-operates with the mitochondrial DNase to facilitate the degradation of kilobase-size DNA, which is followed by oligonucleosome-size DNA laddering.ConclusionsOur results suggest that Tetrahymena AIF plays an important role in the degradation of DNA at an early stage of PND, which supports the notion that the mitochondrion-initiated apoptotic DNA degradation pathway is widely conserved among eukaryotes.
| S-EPMC2829475 | biostudies-literature
Project description:Autoimmune diseases involve a complex dysregulation of immunity. Autoimmune diseases include many members [e.g., rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE)], and most of them are classified according to what organs and tissues are targeted by the damaging immune response. Many studies have focused on finding specific biomarkers for single autoimmune diseases, but so far, there are no universal biomarkers for detecting almost all autoimmune diseases. Serum miRNAs have served as potential biomarkers for detecting various diseases. The purpose of this study was to find a universal biomarker for diagnosing autoimmune diseases. Regulatory T cells (Tregs) play a crucial role in protecting an individual from autoimmunity, and depletion of Tregs in mice is considered a representative animal model of autoimmune disease. Two mouse models for Treg depletion, in which Treg was depleted by CD25mAb (in C57 mice) or by diphtheria toxin (DT) (in Foxp3DTR mice), were investigated, and 381 miRNAs were identified in the serum of mice with Treg depletion. A distinctive circulating miRNA profile was identified in Treg-depleted mice and in patients with autoimmune disease. QRT-PCR confirmation and ROC curve analysis determined that six miRNAs (miR-551b, miR-448, miR-9, miR-124, miR-148, and miR-34c) in the Treg-depleted mouse models and three miRNAs [miR-551b (specificity 73.5%, sensitivity 88.4%), miR-448 (specificity 82.4%, sensitivity 91.3%), and miR-124 (specificity 76.5%, sensitivity 91.3%)] in patients with RA, SLE, Sjogren's syndrome (SS), and ulcerative colitis (UC) could serve as valuable specific biomarkers. These circulating miRNAs may represent potential universal biomarkers for autoimmune diseases diagnosis and prognosis.
| S-EPMC6232775 | biostudies-literature