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Cells Oct 2021To enable long-term survival, mammalian adult neurons exhibit unique apoptosis competence. Questions remain as to whether and how neurons globally reprogram the...
To enable long-term survival, mammalian adult neurons exhibit unique apoptosis competence. Questions remain as to whether and how neurons globally reprogram the expression of apoptotic genes during development. We systematically examined the in vivo expression of 1923 apoptosis-related genes and associated histone modifications at eight developmental ages of mouse brains. Most apoptotic genes displayed consistent temporal patterns across the forebrain, midbrain, and hindbrain, suggesting ubiquitous robust developmental reprogramming. Although both anti- and pro-apoptotic genes can be up- or downregulated, half the regulatory events in the classical apoptosis pathway are downregulation of pro-apoptotic genes. Reduced expression in initiator caspases, apoptosome, and pro-apoptotic Bcl-2 family members restrains effector caspase activation and attenuates neuronal apoptosis. The developmental downregulation of apoptotic genes is attributed to decreasing histone-3-lysine-4-trimethylation (H3K4me3) signals at promoters, where histone-3-lysine-27-trimethylation (H3K27me3) rarely changes. By contrast, repressive H3K27me3 marks are lost in the upregulated gene groups, for which developmental H3K4me3 changes are not predictive. Hence, developing brains remove epigenetic H3K4me3 and H3K27me3 marks on different apoptotic gene groups, contributing to their downregulation and upregulation, respectively. As such, neurons drastically alter global apoptotic gene expression during development to transform apoptosis controls. Research into neuronal cell death should consider maturation stages as a biological variable.
Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Brain; Caspases; Gene Expression Regulation, Developmental; Histone Code; Histones; Lysine; Methylation; Mice; Protein Processing, Post-Translational; Signal Transduction; Time Factors
PubMed: 34831124
DOI: 10.3390/cells10112901 -
Journal of Molecular Biology Feb 2022Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines...
Pyroptosis has been described in mammalian systems to be a form of programmed cell death that is important in immune function through the subsequent release of cytokines and immune effectors upon cell bursting. This form of cell death has been increasingly well-characterized in mammals and can occur using alternative routes however, across phyla, there has been little evidence for the existence of pyroptosis. Here we provide evidence for an ancient origin of pyroptosis in an in vivo immune scenario in Drosophila melanogaster. Crystal cells, a type of insect blood cell, were recruited to wounds and ruptured subsequently releasing their cytosolic content in a caspase-dependent manner. This inflammatory-based programmed cell death mechanism fits the features of pyroptosis, never before described in an in vivo immune scenario in insects and relies on ancient apoptotic machinery to induce proto-pyroptosis. Further, we unveil key players upstream in the activation of cell death in these cells including the apoptosome which may play an alternative role akin to the inflammasome in proto-pyroptosis. Thus, Drosophila may be a suitable model for studying the functional significance of pyroptosis in the innate immune system.
Topics: Animals; Apoptosomes; Caspases; Drosophila melanogaster; Inflammasomes; Mammals; Pyroptosis
PubMed: 34756921
DOI: 10.1016/j.jmb.2021.167333 -
ACS Omega Sep 2021The molecular mechanism of apoptosome activation through conformational changes of Apaf-1 auto-inhibited form remains largely enigmatic. The crystal structure of Apaf-1...
The molecular mechanism of apoptosome activation through conformational changes of Apaf-1 auto-inhibited form remains largely enigmatic. The crystal structure of Apaf-1 suggests that some ionic bonds, including the bond between K192 and D616, are critical for the preservation of the inactive "closed" form of Apaf-1. Here, a split luciferase complementation assay was used to monitor the effect of disrupting this ionic bond on apoptosome activation and caspase-3 activity in cells. The K192E mutation, predicted to disrupt the ionic interaction with D616, increased apoptosome formation and caspase activity, suggesting that this mutation favors the "open"/active form of Apaf-1. However, mutation of D616 to alanine or lysine had different effects. While both mutants favored apoptosome formation such as K192E, D616K cannot activate caspases and D616A activates caspases poorly, and not as well as wild-type Apaf-1. Thus, our data show that the ionic bond between K192 and D616 is critical for maintaining the closed form of Apaf-1 and that disrupting the interaction enhances apoptosome formation. However, our data also reveal that after apoptosome formation, D616 and K192 play a previously unsuspected role in caspase activation. The molecular explanation for this observation is yet to be elucidated.
PubMed: 34514227
DOI: 10.1021/acsomega.1c02274 -
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 -
Cells May 2021Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer...
Exosome-Based Molecular Transfer Activity of Macrophage-Like Cells Involves Viability of Oral Carcinoma Cells: Size Exclusion Chromatography and Concentration Filter Method.
Extracellular vesicles (EV) heterogeneity is a crucial issue in biology and medicine. In addition, tumor-associated macrophages are key components in cancer microenvironment and immunology. We developed a combination method of size exclusion chromatography and concentration filters (SEC-CF) and aimed to characterize different EV types by their size, cargo types, and functions. A human monocytic leukemia cell line THP-1 was differentiated to CD14-positive macrophage-like cells by stimulation with PMA (phorbol 12-myristate 13-acetate) but not M1 or M2 types. Using the SEC-CF method, the following five EV types were fractionated from the culture supernatant of macrophage-like cells: (i) rare large EVs (500-3000 nm) reminiscent of apoptosomes, (ii) EVs (100-500 nm) reminiscent of microvesicles (or microparticles), (iii) EVs (80-300 nm) containing CD9-positive large exosomes (EXO-L), (iv) EVs (20-200 nm) containing unidentified vesicles/particles, and (v) EVs (10-70 nm) containing CD63/HSP90-positive small exosomes (EXO-S) and particles. For a molecular transfer assay, we developed a THP-1-based stable cell line producinga GFP-fused palmitoylation signal (palmGFP) associated with the membrane. The THP1/palmGFP cells were differentiated into macrophages producing palmGFP-contained EVs. The macrophage/palmGFP-secreted EXO-S and EXO-L efficiently transferred the palmGFP to receiver human oral carcinoma cells (HSC-3/palmTomato), as compared to other EV types. In addition, the macrophage-secreted EXO-S and EXO-L significantly reduced the cell viability (ATP content) in oral carcinoma cells. Taken together, the SEC-CF method is useful for the purification of large and small exosomes with higher molecular transfer activities, enabling efficient molecular delivery to target cells.
Topics: Cell Differentiation; Exosomes; Extracellular Vesicles; Humans; Macrophages; Mouth Neoplasms; Tumor Microenvironment; Tumor-Associated Macrophages
PubMed: 34071980
DOI: 10.3390/cells10061328 -
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 -
Current Osteoporosis Reports Jun 2021In this review, we describe the biology of extracellular vesicles (EV) and how they contribute to bone-associated cancers. (Review)
Review
PURPOSE OF REVIEW
In this review, we describe the biology of extracellular vesicles (EV) and how they contribute to bone-associated cancers.
RECENT FINDINGS
Crosstalk between tumor and bone has been demonstrated to promote tumor and metastatic progression. In addition to direct cell-to-cell contact and soluble factors, such as cytokines, EVs mediate crosstalk between tumor and bone. EVs are composed of a heterogenous group of membrane-delineated vesicles of varying size range, mechanisms of formation, and content. These include apoptotic bodies, microvesicles, large oncosomes, and exosomes. EVs derived from primary tumors have been shown to alter bone remodeling and create formation of a pre-metastatic niche that favors development of bone metastasis. Similarly, EVs from marrow stromal cells have been shown to promote tumor progression. Additionally, EVs can act as therapeutic delivery vehicles due to their low immunogenicity and targeting specificity. EVs play critical roles in intercellular communication. Multiple classes of EVs exist based on size on mechanism of formation. In addition to a role in pathophysiology, EVs can be exploited as therapeutic delivery vehicles.
Topics: Bone Neoplasms; Cell Communication; Disease Progression; Extracellular Vesicles; Humans; Signal Transduction; Tumor Microenvironment
PubMed: 33638774
DOI: 10.1007/s11914-021-00668-w -
Cell Death & Disease Feb 2021Execution of the intrinsic apoptotic pathway is controlled by the BCL-2 proteins at the level of the mitochondrial outer membrane (MOM). This family of proteins consists... (Review)
Review
Execution of the intrinsic apoptotic pathway is controlled by the BCL-2 proteins at the level of the mitochondrial outer membrane (MOM). This family of proteins consists of prosurvival (e.g., BCL-2, MCL-1) and proapoptotic (e.g., BIM, BAD, HRK) members, the functional balance of which dictates the activation of BAX and BAK. Once activated, BAX/BAK form pores in the MOM, resulting in cytochrome c release from the mitochondrial intermembrane space, leading to apoptosome formation, caspase activation, and cleavage of intracellular targets. This pathway is induced by cellular stress including DNA damage, cytokine and growth factor withdrawal, and chemotherapy/drug treatment. A well-documented defense of leukemia cells is to shift the balance of the BCL-2 family in favor of the prosurvival proteins to protect against such intra- and extracellular stimuli. Small molecule inhibitors targeting the prosurvival proteins, named 'BH3 mimetics', have come to the fore in recent years to treat hematological malignancies, both as single agents and in combination with standard-of-care therapies. The most significant example of these is the BCL-2-specific inhibitor venetoclax, given in combination with standard-of-care therapies with great success in AML in clinical trials. As the number and variety of available BH3 mimetics increases, and investigations into applying these novel inhibitors to treat myeloid leukemias continue apace the need to evaluate where we currently stand in this rapidly expanding field is clear.
Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Drug Design; Humans; Leukemia, Myeloid; Molecular Mimicry; Molecular Targeted Therapy; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Sulfonamides
PubMed: 33637708
DOI: 10.1038/s41419-021-03500-6 -
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 -
The Journal of Experimental Medicine Mar 2021Podocyte injury is a common hallmark in various glomerular diseases. The level of LRRC55 was increased in podocytes of patients with focal segmental glomerulosclerosis...
Podocyte injury is a common hallmark in various glomerular diseases. The level of LRRC55 was increased in podocytes of patients with focal segmental glomerulosclerosis (FSGS), diabetic nephropathy (DN), and membranous nephropathy (MN). Upregulated LRRC55 and increased intracellular Ca2+ led to BK channel activation and the loss of intracellular potassium, resulting in apoptosome formation and caspase-3 activation in angiotensin II (Ang II)-treated podocytes. Knockout of Lrrc55 or the BK channel prevented the BK current and ameliorated podocyte injury in Ang II-treated mice. Upstream, NFATc3 regulated the expression of LRRC55. Increased LRRC55 expression in podocytes was also evident in animal models of FSGS, DN, and MN. Treatment with losartan or LRRC55 siRNA suppressed LRRC55 expression, prevented BK channel activation, and attenuated podocyte injury in animal models of FSGS, DN, and MN. In conclusion, upregulated LRRC55 promotes BK channel activation and aggravates cell injury in podocytes in FSGS, DN, and MN. LRRC55 inhibition may represent a new therapeutic approach for podocyte injury.
Topics: Angiotensin II; Animals; Apoptosis; Calcium; Cell Nucleus; Humans; Intracellular Space; Ion Channel Gating; Kidney Glomerulus; Large-Conductance Calcium-Activated Potassium Channels; Losartan; Membrane Proteins; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; NFATC Transcription Factors; Podocytes; Potassium; Promoter Regions, Genetic; Protein Transport; TRPC6 Cation Channel; Up-Regulation; Mice
PubMed: 33346797
DOI: 10.1084/jem.20192373