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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 -
The Biochemical Journal Feb 2021Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism...
Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with an exchange between conformational states proposed to regulate changes in function. Here, we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. Three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with the increased overall flexibility of cytochrome c, particularly movement of the Ω loops. Crystal structures of Y48H and G41T complement these studies which overall suggest that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an 'induced fit' mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics. Thus, the mechanism by which the variants increase peroxidase activity is not related to the conformational dynamics of the native hexacoordinate state of cytochrome c. Recent molecular dynamics data proposing conformational mobility of specific cytochrome c regions underpins changes in reduction potential and alkaline transition pK was not fully supported. These data highlight that conformational dynamics of cytochrome c drive some but not all of its properties and activities.
Topics: Amino Acid Substitution; Apoptosis; Apoptosomes; Crystallography, X-Ray; Cytochromes c; Humans; Hydrogen Bonding; Hydrogen Peroxide; Hydrogen-Ion Concentration; Models, Molecular; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Mutation, Missense; Oxidation-Reduction; Point Mutation; Protein Conformation; Recombinant Proteins; Structure-Activity Relationship; U937 Cells
PubMed: 33480393
DOI: 10.1042/BCJ20200793 -
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 -
Biochimie Jan 2021Split luciferase complementary assay has been used to investigate the effect of WD domain deletion on Apaf-1 oligomerization. Apaf-1 is an adaptor molecule in formation...
Split luciferase complementary assay has been used to investigate the effect of WD domain deletion on Apaf-1 oligomerization. Apaf-1 is an adaptor molecule in formation of apoptosome that activates caspase-9, an activation that is a key event in the mitochondrial cell death pathway. Structural studies suggest that normally Apaf-1 is held in an inactive conformation by intramolecular interactions between Apaf-1's nucleotide binding domain and one of its WD40 domains (WD1). In the prevailing model of Apaf-1 activation, cytochrome c binds to sites in WD1 and in Apaf-1's second WD40 domain (WD2), moving WD1 and WD2 closer together and rotating WD1 away from the nucleotide binding domain. This allows Apaf-1 to bind dATP or ATP and to form the apoptosome, which activates caspase-9. This model predicts that cytochrome c binding to both WD domains is necessary for apoptosome formation and that an Apaf-1 with only WD1 will be locked in an inactive conformation that cannot be activated by cytochrome c. Here we investigated the effect of removing one WD domain (Apaf-1 1-921) on Apaf-1 interactions and caspase activation. Apaf-1 1-921 could not activate caspase-9, even in the presence of cytochrome c. These data show that a single WD domain is sufficient to lock Apaf-1 in an inactive state and this state cannot be altered by cytochrome c.
Topics: Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 9; Cytochromes c; Deoxyadenine Nucleotides; Enzyme Activation; HEK293 Cells; Humans; Luciferases; Luminescent Measurements; Mutation; Protein Binding; Protein Structure, Quaternary; Recombinant Proteins; WD40 Repeats
PubMed: 33132160
DOI: 10.1016/j.biochi.2020.10.013 -
Cell Death & Disease Sep 2020Intrinsic apoptosis as a modality of regulated cell death is intimately linked to permeabilization of the outer mitochondrial membrane and subsequent release of the...
Intrinsic apoptosis as a modality of regulated cell death is intimately linked to permeabilization of the outer mitochondrial membrane and subsequent release of the protein cytochrome c into the cytosol, where it can participate in caspase activation via apoptosome formation. Interestingly, cytochrome c release is an ancient feature of regulated cell death even in unicellular eukaryotes that do not contain an apoptosome. Therefore, it was speculated that cytochrome c release might have an additional, more fundamental role for cell death signalling, because its absence from mitochondria disrupts oxidative phosphorylation. Here, we permanently anchored cytochrome c with a transmembrane segment to the inner mitochondrial membrane of the yeast Saccharomyces cerevisiae, thereby inhibiting its release from mitochondria during regulated cell death. This cytochrome c retains respiratory growth and correct assembly of mitochondrial respiratory chain supercomplexes. However, membrane anchoring leads to a sensitisation to acetic acid-induced cell death and increased oxidative stress, a compensatory elevation of cellular oxygen-consumption in aged cells and a decreased chronological lifespan. We therefore conclude that loss of cytochrome c from mitochondria during regulated cell death and the subsequent disruption of oxidative phosphorylation is not required for efficient execution of cell death in yeast, and that mobility of cytochrome c within the mitochondrial intermembrane space confers a fitness advantage that overcomes a potential role in regulated cell death signalling in the absence of an apoptosome.
Topics: Cell Death; Cytochromes c; Humans; Mitochondria; Yeasts
PubMed: 32892209
DOI: 10.1038/s41419-020-02920-0 -
Communications Biology Aug 2020Early studies of the free-living nematode C. elegans informed us how BCL-2-regulated apoptosis in humans is regulated. However, subsequent studies showed C. elegans...
Early studies of the free-living nematode C. elegans informed us how BCL-2-regulated apoptosis in humans is regulated. However, subsequent studies showed C. elegans apoptosis has several unique features compared with human apoptosis. To date, there has been no detailed analysis of apoptosis regulators in nematodes other than C. elegans. Here, we discovered BCL-2 orthologues in 89 free-living and parasitic nematode taxa representing four evolutionary clades (I, III, IV and V). Unlike in C. elegans, 15 species possess multiple (two to five) BCL-2-like proteins, and some do not have any recognisable BCL-2 sequences. Functional studies provided no evidence that BAX/BAK proteins have evolved in nematodes, and structural studies of a BCL-2 protein from the basal clade I revealed it lacks a functionally important feature of the C. elegans orthologue. Clade I CED-4/APAF-1 proteins also possess WD40-repeat sequences associated with apoptosome assembly, not present in C. elegans, or other nematode taxa studied.
Topics: Animals; Apoptosis; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Genes, Helminth; Mice; Phylogeny; Protein Domains; Protein Structure, Secondary; Proto-Oncogene Proteins c-bcl-2; Signal Transduction
PubMed: 32859965
DOI: 10.1038/s42003-020-01208-5 -
Cellular Signalling Nov 2020Anaplastic thyroid cancer (ATC) is a rare malignancy and has a poor prognosis due to its aggressive behavior and resistance to treatments. Calcium (Ca) serves as a...
Anaplastic thyroid cancer (ATC) is a rare malignancy and has a poor prognosis due to its aggressive behavior and resistance to treatments. Calcium (Ca) serves as a ubiquitous cellular second messenger and influences several tumor behaviors. Therefore, Ca modulation is expected to be a novel therapeutic target in cancers. However, whether Ca modulation is effective in ATC therapy remains unknown. In this study, we reported that capsaicin (CAP), a transient receptor potential vanilloid type1 (TRPV1) agonist, inhibited the viability of anaplastic thyroid cancer cells. Capsaicin treatment triggered Ca influx by TRPV1 activation, resulting in disequilibrium of intracellular calcium homeostasis. The rapidly increased cytosolic Ca concentration was mirrored in the mitochondria and caused a severe condition of mitochondrial calcium overload in ATC cells. In addition, the disruption of mitochondrial calcium homeostasis caused by capsaicin led to mitochondrial dysfunction in ATC cells, as evidenced by the production of mitochondrial reactive oxygen species (ROS), depolarization of mitochondrial membrane potential (ΔΨm), and opening of mitochondrial permeability transition pore (mPTP). Next, the resulting release of cyt c into the cytosol triggered apoptosome assembly and subsequent caspase activation and apoptosis. It was worth noting that both TRPV1 antagonist (capsazepine) and calcium chelator (BAPTA) could attenuate aberrant Ca homeostasis, mitochondrial dysfunction and apoptosis induced by capsaicin treatment. Thus, our study demonstrated that capsaicin induced mitochondrial calcium overload and apoptosis in ATC cells through a TRPV1-mediated pathway. The better understanding of the anti-cancer mechanisms of calcium modulation provides a potential target for the ATC therapy.
Topics: Antineoplastic Agents; Apoptosis; Calcium; Capsaicin; Cell Line, Tumor; Humans; Mitochondria; Thyroid Carcinoma, Anaplastic; Thyroid Neoplasms
PubMed: 32771398
DOI: 10.1016/j.cellsig.2020.109733 -
Apoptosis : An International Journal on... Oct 2020Apoptosis plays a major role in development, tissue renewal and the progression of degenerative diseases. Studies on various types of mammalian cells reported a...
Apoptosis plays a major role in development, tissue renewal and the progression of degenerative diseases. Studies on various types of mammalian cells reported a pro-apoptotic function of acetylcholinesterase (AChE), particularly in the formation of the apoptosome and the degradation of nuclear DNA. While three AChE splice variants are present in mammals, invertebrates typically express two ache genes that code for a synaptically located protein and a protein with non-synaptic functions respectively. In order to investigate a potential contribution of AChE to apoptosis in insects, we selected the migratory locust Locusta migratoria. We established primary neuronal cultures of locust brains and characterized apoptosis progression in vitro. Dying neurons displayed typical characteristics of apoptosis, including caspase-activation, nuclear condensation and DNA fragmentation visualized by TUNEL staining. Addition of the AChE inhibitors neostigmine and territrem B reduced apoptotic cell death under normal culture conditions. Moreover, both inhibitors completely suppressed hypoxia-induced neuronal cell death. Exposure of live animals to severe hypoxia moderately increased the expression of ace-1 in locust brains in vivo. Our results indicate a previously unreported role of AChE in insect apoptosis that parallels the pro-apoptotic role in mammalian cells. This similarity adds to the list of apoptotic mechanisms shared by mammals and insects, supporting the hypothesized existence of an ancient, complex apoptosis regulatory network present in common ancestors of vertebrates and insects.
Topics: Acetylcholinesterase; Animals; Apoptosis; Brain; Cell Death; Cell Nucleus; DNA Fragmentation; Grasshoppers; Hypoxia; Insecta; Neurons; Peptidyl-Dipeptidase A
PubMed: 32761307
DOI: 10.1007/s10495-020-01630-4 -
Development (Cambridge, England) Jul 2020Cell death is an important facet of animal development. In some developing tissues, death is the ultimate fate of over 80% of generated cells. Although recent studies... (Review)
Review
Cell death is an important facet of animal development. In some developing tissues, death is the ultimate fate of over 80% of generated cells. Although recent studies have delineated a bewildering number of cell death mechanisms, most have only been observed in pathological contexts, and only a small number drive normal development. This Primer outlines the important roles, different types and molecular players regulating developmental cell death, and discusses recent findings with which the field currently grapples. We also clarify terminology, to distinguish between developmental cell death mechanisms, for which there is evidence for evolutionary selection, and cell death that follows genetic, chemical or physical injury. Finally, we suggest how advances in understanding developmental cell death may provide insights into the molecular basis of developmental abnormalities and pathological cell death in disease.
Topics: Animals; Apoptosis; Apoptosomes; Autophagy; Caspases; Cell Death; Gene Expression Regulation, Developmental; Invertebrates; Mammals; Signal Transduction
PubMed: 32709690
DOI: 10.1242/dev.191882