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Redox Biology Nov 2022Nutritional pancreatic atrophy (NPA) is a classical Se/vitamin E deficiency disease of chicks. To reveal molecular mechanisms of its pathogenesis, we fed day-old chicks...
Nutritional pancreatic atrophy (NPA) is a classical Se/vitamin E deficiency disease of chicks. To reveal molecular mechanisms of its pathogenesis, we fed day-old chicks a practical, low-Se diet (14 μg Se/kg), and replicated the typical symptoms of NPA including vesiculated mitochondria, cytoplasmic vacuoles, and hyaline bodies in acinar cells of chicks as early as day 18. Target pathway analyses illustrated a > 90% depletion (P < 0.05) of glutathione peroxidase 4 (GPX4) protein and up-regulated apoptotic signaling (cytochrome C/caspase 9/caspase 3) in the pancreas and(or) acinar cells of Se deficient chicks compared with Se-adequate chicks. Subsequently, we overexpressed and suppressed GPX4 expression in the pancreatic acinar cells and observed an inverse (P < 0.05) relationship between the GPX4 production and apoptotic signaling and cell death. Applying pull down and mass spectrometry, we unveiled that GPX4 bound prothymosin alpha (ProTalpha) to inhibit formation of apoptosome in the pancreatic acinar cells. Destroying this novel protein-protein interaction by silencing either gene expression accelerated HO-induced apoptosis in the cells. In the end, we applied GPX4 shRNA to silence GPX4 expression in chick embryo and confirmed the physiological relevance of the GPX4 role and mechanism shown ex vivo and in the acinar cells. Altogether, our results indicated that GPX4 depletion in Se-deficient chicks acted as a major contributor to their development of NPA due to the lost binding of GPX4 to ProTalpha and its subsequent inhibition on the cytochrome c/caspase 9/caspase 3 cascade in the acinar cells. Our findings not only provide a novel molecular mechanism for explaining pathogenesis of NPA but also reveal a completely new cellular pathway in regulating apoptosis by selenoproteins.
PubMed: 36162257
DOI: 10.1016/j.redox.2022.102482 -
Cellular and Molecular Neurobiology Jul 2022Targeting apoptosis in the ischemic penumbra is a rational therapeutic approach for restricting cerebral infarct volume after clinical stroke. The present work explored...
Targeting apoptosis in the ischemic penumbra is a rational therapeutic approach for restricting cerebral infarct volume after clinical stroke. The present work explored the capability of the obestatin peptide, as a novel approach to inhibit apoptotic signaling cascades on PC12 cells. According to the results, obestatin treatment significantly reduced nutrient deprivation-induced apoptotic cell death. The protective effects were related to the regulation of the anti-apoptotic protein, BCL-2, and the apoptotic protein caspase-3. This encompasses the control of apoptosis by the interplay between Akt, ERK1/2 and AMPK signaling pathways. The activation of Akt and AMPK was concomitant with the phosphorylation of their downstream targets, GSK3 and ACC, respectively. Besides, obestatin also causes FoxO1 nuclear export supporting the prevention of the apoptosome formation. The concurrent activation of Akt and AMPK by obestatin via the GPR39 receptor, supports a role for this system in the balance concerning the catabolic and the anabolic signaling to sustain cellular function and viability. Furthermore, these results provide both an insight into how the obestatin/GPR39 system regulates anti-apoptotic pathways, and a framework for ascertaining how this system can be optimally targeted in treatment of brain cell death after stroke.
Topics: AMP-Activated Protein Kinases; Animals; Apoptosis; Ghrelin; Glycogen Synthase Kinase 3; MAP Kinase Signaling System; Nutrients; PC12 Cells; Proto-Oncogene Proteins c-akt; Rats; Receptors, G-Protein-Coupled; Stroke
PubMed: 33400083
DOI: 10.1007/s10571-020-01025-8 -
Disease Markers 2022Lung adenocarcinoma (LUAD) is the most common subtype of nonsmall cell lung cancer. Cytochrome c (Cyt c), which is produced from mitochondria, interacts with a protein...
Lung adenocarcinoma (LUAD) is the most common subtype of nonsmall cell lung cancer. Cytochrome c (Cyt c), which is produced from mitochondria, interacts with a protein called Apaf-1 to form the heptameric apoptosome. This heptameric apoptosome then activates the caspase cascade, which ultimately results in the execution of apoptosis. The purpose of our research was to discover a new prognostic model that is based on cytochrome c-related genes (CCRGs) for LUAD patients. Through LASSO regression analysis conducted on the LUAD datasets included in the TCGA datasets, a CCRGs signature was created. The diagnostic accuracy of the multigene signature was verified by an independent source using the GSE31210 and GSE72094 datasets. The GO and KEGG enrichment analysis were performed. In this study, there were 159 differentially expressed CCRGs in the TCGA dataset, while there were 68 differentially expressed CCRGs in the GSE31210 dataset. Additionally, there were 57 genes that overlapped across the two datasets. Using LASSO and Cox regression analysis, a signature consisting of 12 differentially expressed CCRGs was developed from the total of 57 such genes. On the basis of their risk ratings, patients were categorized into high-risk and low-risk categories, with low-risk patients having lower risk scores and a greater likelihood of surviving the disease. Univariate and multivariate analyses both concluded that this signature is an independent risk factor for LUAD. ROC curves demonstrated that this risk signature is capable of accurately predicting the 1-year, 2-year, 3-year, and 5-year survival rates of patients who have LUAD. The infiltration of antigen-presenting cells was higher in the low-risk group, such as aDCs, DCs, pDCs, and iDCs. The expression of multiple immune checkpoints was significantly higher in the low-risk group, such as BTLA, CD28, and CD86. Finally, we showed that the signature can be used to predict the drug sensitivity of already available or under investigational drugs. Overall, patient classification and individualized therapy options may benefit from this study's development of a powerful gene signature with high value for prognostic prediction in LUAD.
Topics: Adenocarcinoma of Lung; Apoptosomes; CD28 Antigens; Carcinoma, Non-Small-Cell Lung; Caspases; Cytochromes c; Drugs, Investigational; Humans; Lung Neoplasms; Prognosis
PubMed: 36225197
DOI: 10.1155/2022/8809956 -
Angewandte Chemie (International Ed. in... May 2021Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase-9, using short...
Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase-9, using short DNA elements as modular recruitment domains. Caspase-9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase-9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.
Topics: Caspase 9; DNA; Humans; Kinetics; Models, Molecular
PubMed: 33725379
DOI: 10.1002/anie.202102160 -
Toxins Jan 2020T-2 toxin is type A trichothecenes mycotoxin, which produced by fusarium species in cereal grains. T-2 toxin has been shown to induce a series of toxic effects on the...
T-2 toxin is type A trichothecenes mycotoxin, which produced by fusarium species in cereal grains. T-2 toxin has been shown to induce a series of toxic effects on the health of human and animal, such as immunosuppression and carcinogenesis. Previous study has proven that T-2 toxin caused hepatotoxicity in chicken, but the regulatory mechanism is unclear. In the present study, we assessed the toxicological effect of T-2 toxin on apoptosis and autophagy in hepatocytes. The total of 120 1-day-old healthy broilers were allocated randomly into four groups and reared for 21 day with complete feed containing 0 mg/kg, 0.5 mg/kg, 1 mg/kg or 2 mg/kg T-2 toxin, respectively. The results showed that the apoptosis rate and pathological changes degree hepatocytes were aggravated with the increase of T-2 toxin. At the molecular mechanism level, T-2 toxin induced mitochondria-mediated apoptosis by producing reactive oxygen species, promoting cytochrome c translocation between the mitochondria and cytoplasm, and thus promoting apoptosomes formation. Meanwhile, the expression of the autophagy-related protein, ATG5, ATG7 and Beclin-1, and the LC3-II/LC3-I ratio were increased, while p62 was downregulated, suggesting T-2 toxin caused autophagy in hepatocytes. Further experiments demonstrated that the PI3K/AKT/mTOR signal may be participated in autophagy induced by T-2 toxin in chicken hepatocytes. These data suggest a possible underlying molecular mechanism for T-2 toxin that induces apoptosis and autophagy in chicken hepatocytes.
Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Autophagy-Related Proteins; Chickens; Hepatocytes; Liver; Male; Mitochondria; Oxidative Stress; Reactive Oxygen Species; T-2 Toxin
PubMed: 32013230
DOI: 10.3390/toxins12020090 -
Cell Death and Differentiation Oct 2020The execution phase of apoptosis is a critical process in programmed cell death in response to a multitude of cellular stresses. A crucial component of this pathway is...
The execution phase of apoptosis is a critical process in programmed cell death in response to a multitude of cellular stresses. A crucial component of this pathway is the apoptosome, a platform for the activation of pro-caspase 9 (PC9). Recent findings have shown that autocleavage of PC9 to Caspase 9 (C9) p35/p12 not only permits XIAP-mediated C9 inhibition but also temporally shuts down apoptosome activity, forming a molecular timer. In order to delineate the combined contributions of XIAP and the apoptosome molecular timer to apoptosis execution we utilised a systems modelling approach. We demonstrate that cooperative recruitment of PC9 to the apoptosome, based on existing PC9-apoptosome interaction data, is important for efficient formation of PC9 homodimers, autocatalytic cleavage and dual regulation by XIAP and the molecular timer across biologically relevant PC9 and APAF1 concentrations. Screening physiologically relevant concentration ranges of apoptotic proteins, we discovered that the molecular timer can prevent apoptosis execution in specific scenarios after complete or partial mitochondrial outer membrane permeabilisation (MOMP). Furthermore, its ability to prevent apoptosis is intricately tied to a synergistic combination with XIAP. Finally, we demonstrate that simulations of these processes are prognostic of survival in stage III colorectal cancer and that the molecular timer may promote apoptosis resistance in a subset of patients. Based on our findings, we postulate that the physiological function of the molecular timer is to aid XIAP in the shutdown of caspase-mediated apoptosis execution. This shutdown potentially facilitates switching to pro-inflammatory caspase-independent responses subsequent to Bax/Bak pore formation.
Topics: Apoptosis; Caspase 9; Colorectal Neoplasms; Humans; Mitochondria; Mitochondrial Membranes; X-Linked Inhibitor of Apoptosis Protein
PubMed: 32341447
DOI: 10.1038/s41418-020-0545-9 -
Nature Catalysis Mar 2020Living cells regulate key cellular processes by spatial organisation of catalytically active proteins in higher-order signalling complexes. These act as organising...
Living cells regulate key cellular processes by spatial organisation of catalytically active proteins in higher-order signalling complexes. These act as organising centres to facilitate proximity-induced activation and inhibition of multiple intrinsically weakly associating signalling components, which makes elucidation of the underlying protein-protein interactions challenging. Here we show that DNA origami nanostructures provide a programmable molecular platform for the systematic analysis of signalling proteins by engineering a synthetic DNA origami-based version of the apoptosome, a multi-protein complex that regulates apoptosis by co-localizing multiple caspase-9 monomers. Tethering of both wildtype and inactive caspase-9 variants to a DNA origami platform demonstrates that enzymatic activity is induced by proximity-driven dimerization with half-of-sites reactivity, and additionally, reveals a multivalent activity enhancement in oligomers of three and four enzymes. Our results offer fundamental insights in caspase-9 activity regulation and demonstrate that DNA origami-based protein assembly platforms have the potential to inform the function of other multi-enzyme complexes involved in inflammation, innate immunity and cell death.
PubMed: 32190819
DOI: 10.1038/s41929-019-0403-7 -
The International Journal of... Apr 2020Cytochrome c (Cytc)is a cellular life and death decision molecule that regulates cellular energy supply and apoptosis through tissue specific post-translational...
Cytochrome c (Cytc)is a cellular life and death decision molecule that regulates cellular energy supply and apoptosis through tissue specific post-translational modifications. Cytc is an electron carrier in the mitochondrial electron transport chain (ETC) and thus central for aerobic energy production. Under conditions of cellular stress, Cytc release from the mitochondria is a committing step for apoptosis, leading to apoptosome formation, caspase activation, and cell death. Recently, Cytc was shown to be a target of cellular signaling pathways that regulate the functions of Cytc by tissue-specific phosphorylations. So far five phosphorylation sites of Cytc have been mapped and functionally characterized, Tyr97, Tyr48, Thr28, Ser47, and Thr58. All five phosphorylations partially inhibit respiration, which we propose results in optimal intermediate mitochondrial membrane potentials and low ROS production under normal conditions. Four of the phosphorylations result in inhibition of the apoptotic functions of Cytc, suggesting a cytoprotective role for phosphorylated Cytc. Interestingly, these phosphorylations are lost during stress conditions such as ischemia. This results in maximal ETC flux during reperfusion, mitochondrial membrane potential hyperpolarization, excessive ROS generation, and apoptosis. We here present a new model proposing that the electron transfer from Cytc to cytochrome c oxidase is the rate-limiting step of the ETC, which is regulated via post-translational modifications of Cytc. This regulation may be dysfunctional in disease conditions such as ischemia-reperfusion injury and neurodegenerative disorders through increased ROS, or cancer, where post-translational modifications on Cytc may provide a mechanism to evade apoptosis.
Topics: Apoptosis; Cytochromes c; Electron Transport; Humans; Phosphorylation
PubMed: 32023432
DOI: 10.1016/j.biocel.2020.105704 -
Cells Mar 2024Cytochrome (Cyt) has both life-sustaining and cellular death-related functions, depending on subcellular localization. Within mitochondria, Cyt acts as a single... (Review)
Review
Cytochrome (Cyt) has both life-sustaining and cellular death-related functions, depending on subcellular localization. Within mitochondria, Cyt acts as a single electron carrier as part of the electron transport chain (ETC). When released into the cytosol after cellular insult, Cyt triggers the assembly of the apoptosome, committing the cell to intrinsic apoptosis. Due to these dual natures, Cyt requires strong regulation by the cell, including post-translational modifications, such as phosphorylation and acetylation. Six phosphorylation sites and three acetylation sites have been detected on Cyt in vivo. Phosphorylations at T28, S47, Y48, T49, T58, and Y97 tend to be present under basal conditions in a tissue-specific manner. In contrast, the acetylations at K8, K39, and K53 tend to be present in specific pathophysiological conditions. All of the phosphorylation sites and two of the three acetylation sites partially inhibit respiration, which we propose serves to maintain an optimal, intermediate mitochondrial membrane potential (ΔΨ) to minimize reactive oxygen species (ROS) production. Cyt phosphorylations are lost during ischemia, which drives ETC hyperactivity and ΔΨ hyperpolarization, resulting in exponential ROS production thus causing reperfusion injury following ischemia. One of the acetylation sites, K39, shows a unique behavior in that it is gained during ischemia, stimulating respiration while blocking apoptosis, demonstrating that skeletal muscle, which is particularly resilient to ischemia-reperfusion injury compared to other organs, possesses a different metabolic strategy to handle ischemic stress. The regulation of Cyt by these post-translational modifications underscores the importance of Cyt for the ETC, ΔΨ, ROS production, apoptosis, and the cell as a whole.
Topics: Humans; Phosphorylation; Cytochromes c; Acetylation; Reactive Oxygen Species; Membrane Potential, Mitochondrial; Mitochondria; Apoptosis; Respiration; Ischemia
PubMed: 38534337
DOI: 10.3390/cells13060493 -
Biochemical and Biophysical Research... Nov 2021Liver cancer is one of the most common malignancies that is difficult to treat due to late diagnosis and chemo-resistance. In the present study, we developed and...
Liver cancer is one of the most common malignancies that is difficult to treat due to late diagnosis and chemo-resistance. In the present study, we developed and validated a cell based split nanoLuc biosensor to monitor the Apaf1-Apaf1 interactions in response to apoptosis-inducing drugs such as cisplatin. We showed that the activity of split nanoLuc is reconstituted only in response to apoptotic inducer, cisplatin and in a dose-dependent manner. Apaf1 mutants which were unable to oligomerize failed to recover nanoLuc activity while constitutively active variant increased the nanoLuc activity. Generation of Apaf1 knockout HepG2 and treatment with cisplatin showed dramatic reduction in cell death suggesting that cisplatin mainly targets liver cancer cells through apoptosis. As the natural products are potent sources of compounds for adjuvant therapy, we screened a collection of natural products and identified lentinan as an inducer of apoptosome formation, a key step for induction of apoptosis. Lentinan is a polysaccharide with antitumor, pro-apoptotic properties that functions with poorly understood mechanisms. Lentinan was shown to have cytotoxic effects with the IC of 650 μM. Sub-lethal lentinan concentration doubled the nanoLuc activity when co-treated with cisplatin. We also showed that lentinan hugely reduced the dose of cisplatin to induce certain amount of death and that lentinan co-treatment with cisplatin enhanced the Apaf1 transcription in HepG2 cells while lentinan or cisplatin alone failed to alter the transcription. In addition, lentinan and cisplatin co-treatment induced mitochondrial depolarization. This suggested that lentinan combinatorial therapy with cisplatin engaged a different signalling pathway to kill the liver cancer cells and that adjuvant therapy with lentinan can reduce the dose of cisplatin and thus reduce the possibility of chemo-resistance.
Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Apoptotic Protease-Activating Factor 1; Biosensing Techniques; Carcinoma, Hepatocellular; Cisplatin; Drug Synergism; Hep G2 Cells; Humans; Lentinan; Liver Neoplasms; Mutation
PubMed: 34507064
DOI: 10.1016/j.bbrc.2021.08.030