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Experimental and Therapeutic Medicine Jan 2018Avoidance of apoptosis induced by anticancer drugs is an essential factor of carcinogenesis and a hallmark of resistance to cancer therapy. Human telomerase reverse...
Avoidance of apoptosis induced by anticancer drugs is an essential factor of carcinogenesis and a hallmark of resistance to cancer therapy. Human telomerase reverse transcriptase receptor (hTERTR) is a potential anti-cancer agent for inhibiting tumor growth. Family with sequence similarity 96 member A (FAM96A) is a ubiquitous, conserved protein and possesses apoptosome-activating and pro-apoptotic tumor suppressor potential in hepatocellular carcinoma (HCC). In the present study, hTERTR and FAM96A were identified as efficient anti-cancer agents for activating apoptosomes and reducing tumor growth. The potential tumor suppressor function of combination treatment with hTERTR and FAM96A in HCC was also investigated. hTERTR and FAM96A proteins were expressed by genetic engineering and their anti-cancer function was explored and . Effects of hTERTR and FAM96A on improvement of apoptotic sensitivity and inhibition of migration and invasion were examined in cancer cells and in a mouse model. The present results demonstrated that the therapeutic effects of hTERTR and FAM96A were effective for inhibiting tumor growth and inducing apoptosis of HCC cells in H22-bearing nude mice compared with single agent treatment. hTERTR and FAM96A were found to bind with apoptotic protease activating factor 1 and human telomerase reverse transcriptase, which enhanced the apoptosis of tumor cells and apoptosis sensitivity. In addition, hTERTR and FAM96A therapy enhanced cytotoxic effects by cytotoxic T lymphocyte responses, interferon-γ release, T lymphocytes infiltration and apoptosis on tumor cells. Furthermore, hTERTR and FAM96A protein inhibited tumor growth in HCC mice. In conclusion, the present findings suggested that combination therapy with hTERTR and FAM96A may serve as novel tumor suppressor agents.
PubMed: 29399066
DOI: 10.3892/etm.2017.5505 -
Proceedings of the National Academy of... Feb 2017Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The...
Mammalian intrinsic apoptosis requires activation of the initiator caspase-9, which then cleaves and activates the effector caspases to execute cell killing. The heptameric Apaf-1 apoptosome is indispensable for caspase-9 activation by together forming a holoenzyme. The molecular mechanism of caspase-9 activation remains largely enigmatic. Here, we report the cryoelectron microscopy (cryo-EM) structure of an apoptotic holoenzyme and structure-guided biochemical analyses. The caspase recruitment domains (CARDs) of Apaf-1 and caspase-9 assemble in two different ways: a 4:4 complex docks onto the central hub of the apoptosome, and a 2:1 complex binds the periphery of the central hub. The interface between the CARD complex and the central hub is required for caspase-9 activation within the holoenzyme. Unexpectedly, the CARD of free caspase-9 strongly inhibits its proteolytic activity. These structural and biochemical findings demonstrate that the apoptosome activates caspase-9 at least in part through sequestration of the inhibitory CARD domain.
Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 9; Caspase Activation and Recruitment Domain; Cryoelectron Microscopy; Enzyme Activation; Holoenzymes; Humans; Models, Molecular; Mutation; Protein Binding; Protein Domains; Protein Multimerization
PubMed: 28143931
DOI: 10.1073/pnas.1620626114 -
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 -
International Journal of Molecular... Sep 2023Virus-specific proteins, including coat proteins, movement proteins, replication proteins, and suppressors of RNA interference are capable of triggering the... (Review)
Review
Virus-specific proteins, including coat proteins, movement proteins, replication proteins, and suppressors of RNA interference are capable of triggering the hypersensitive response (HR), which is a type of cell death in plants. The main cell death signaling pathway involves direct interaction of HR-inducing proteins with nucleotide-binding leucine-rich repeats (NLR) proteins encoded by plant resistance genes. Singleton NLR proteins act as both sensor and helper. In other cases, NLR proteins form an activation network leading to their oligomerization and formation of membrane-associated resistosomes, similar to metazoan inflammasomes and apoptosomes. In resistosomes, coiled-coil domains of NLR proteins form Ca channels, while toll-like/interleukin-1 receptor-type (TIR) domains form oligomers that display NAD+ glycohydrolase (NADase) activity. This review is intended to highlight the current knowledge on plant innate antiviral defense signaling pathways in an attempt to define common features of antiviral resistance across the kingdoms of life.
Topics: Animals; Signal Transduction; Antiviral Agents; Hypersensitivity; NLR Proteins; Phagocytosis; Viruses
PubMed: 37686431
DOI: 10.3390/ijms241713625 -
Nature Communications Nov 2016According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form...
According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.
Topics: Animals; Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Caspase 3; Caspase 9; Dimerization; Mice; Phosphotransferases; Protein Kinases; Sf9 Cells; Spodoptera; Tumor Suppressor Proteins
PubMed: 27882936
DOI: 10.1038/ncomms13565 -
Autophagy Jun 2021Recently, we reported that increased expression of CASP9 pro-domain, at the endosomal membrane in response to HSP90 inhibition, mediates a cell-protective effect that...
Recently, we reported that increased expression of CASP9 pro-domain, at the endosomal membrane in response to HSP90 inhibition, mediates a cell-protective effect that does not involve CASP9 apoptotic activity. We report here that a non-apoptotic activity of endosomal membrane CASP9 facilitates the retrograde transport of IGF2R/CI-MPR from the endosomes to the trans-Golgi network, indicating the involvement of CASP9 in endosomal sorting and lysosomal biogenesis. CASP9-deficient cells demonstrate the missorting of CTSD (cathepsin D) and other acid hydrolases, accumulation of late endosomes, and reduced degradation of bafilomycin A-sensitive proteins. In the absence of CASP9, IGF2R undergoes significant degradation, and its rescue is achieved by the re-expression of a non-catalytic mutant. This endosomal activity of CASP9 is potentially mediated by herein newly identified interactions of CASP9 with the components of the endosomal membrane transport complexes. These endosomal complexes include the retromer VPS35 and the SNX dimers, SNX1-SNX5 and SNX2-SNX6, which are involved in the IGF2R retrieval mechanism. Additionally, CASP9 interacts with HGS/HRS/ESCRT-0 and the CLTC (clathrin heavy chain) that participate in the initiation of the endosomal ESCRT degradation pathway. We propose that endosomal CASP9 inhibits the endosomal membrane degradative subdomain(s) from initiating the ESCRT-mediated degradation of IGF2R, allowing its retrieval to transport-designated endosomal membrane subdomain(s). These findings are the first to identify a cell survival, non-apoptotic function for CASP9 at the endosomal membrane, a site distinctly removed from the cytoplasmic apoptosome. Via its non-apoptotic endosomal function, CASP9 impacts the retrograde transport of IGF2R and, consequently, lysosomal biogenesis.: ACTB: actin beta; ATG7: autophagy related 7; BafA1: bafilomycin A; CASP: caspase; CLTC/CHC: clathrin, heavy chain; CTSD: cathepsin D; ESCRT: endosomal sorting complexes required for transport; HEXB: hexosaminidase subunit beta; HGS/HRS/ESCRT-0: hepatocyte growth factor-regulated tyrosine kinase substrate; IGF2R/CI-MPR: insulin like growth factor 2 receptor; ILV: intraluminal vesicles; KD: knockdown; KO: knockout; M6PR/CD-MPR: mannose-6-phosphate receptor, cation dependent; MEF: murine embryonic fibroblasts; MWU: Mann-Whitney U test; PepA: pepstatin A; RAB7A: RAB7, member RAS oncogene family; SNX-BAR: sorting nexin dimers with a Bin/Amphiphysin/Rvs (BAR) domain each; TGN: trans-Golgi network; TUBB: tubulin beta; VPS26: VPS26 retromer complex component; VPS29: VPS29 retromer complex component; VPS35: VPS35 retromer complex component.
Topics: Autophagy; Caspase 9; Endosomes; HeLa Cells; Humans; Microtubule-Associated Proteins; Protein Transport; Receptor, IGF Type 2; Vesicular Transport Proteins; trans-Golgi Network
PubMed: 32397873
DOI: 10.1080/15548627.2020.1761742 -
Food and Chemical Toxicology : An... Jul 2023The present study aims to investigate the role of liensinine in life-threatened sepsis-associated encephalopathy (SAE) mice and the underlying mechanism. Here,...
Liensinine, a alkaloid from lotus plumule, mitigates lipopolysaccharide-induced sepsis-associated encephalopathy through modulation of nuclear factor erythroid 2-related factor-mediated inflammatory biomarkers and mitochondria apoptosis.
The present study aims to investigate the role of liensinine in life-threatened sepsis-associated encephalopathy (SAE) mice and the underlying mechanism. Here, seventy-two mice were divided into six groups, including the control group, SAE group, liensinine-treated group, and three doses of liensinine-treated SAE groups. Lipopolysaccharide triggered cerebrum necrosis and disrupted the integrity and permeability of blood-brain barrier (BBB). While liensinine restored cerebrum structure and improved BBB integrity with upregulated tight junction proteins, decreased evans blue leakage and fibrinogen expression with decreased matrix metalloproteinases 2/9 in serum, thereby reducing BBB permeability. Moreover, lipopolysaccharide triggered cerebrum oxidative stress and inflammation, whereas liensinine enhanced antioxidant enzymes activities and weakened malondialdehyde through nuclear factor erythroid 2-related factor. Meanwhile, liensinine inhibited inflammation by activating inducible nitric oxide synthase. Tunel staining combined with transmission electron microscope indicated that lipopolysaccharide induced cerebrum apoptosis, whereas liensinine blocked apoptosis through decreasing B-cell lymphoma-2 associated X (Bax) expression and cytochrome C (Cyto-c) release, increasing B-cell lymphoma-2 (Bcl-2) expression, blocking apoptosome assembly, inhibiting caspase-3 activation, thereby suppressing intrinsic mitochondria apoptosis. Recovering of inflammatory homeostasis and inhibition of mitochondria apoptosis by liensinine ultimately restored cognitive function in SAE mice. Altogether, liensinine attenuated lipopolysaccharide-induced SAE via modulation of Nrf2-mediated inflammatory biomarkers and mitochondria apoptosis.
Topics: Mice; Animals; Sepsis-Associated Encephalopathy; Lipopolysaccharides; Lotus; Apoptosis; Antineoplastic Agents; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Inflammation; Alkaloids
PubMed: 37150347
DOI: 10.1016/j.fct.2023.113813 -
Current Issues in Molecular Biology Nov 2021Hepatitis C virus (HCV)-induced liver disease contributes to chronic hepatitis. The immune factors identified in HCV include changes in the innate and adaptive immune...
Hepatitis C virus (HCV)-induced liver disease contributes to chronic hepatitis. The immune factors identified in HCV include changes in the innate and adaptive immune system. The inflammatory mediators, known as "inflammasome", are a consequence of the metabolic products of cells and commensal or pathogenic bacteria and viruses. The only effective strategy to prevent disease progression is eradication of the viral infection. Immune cells play a pivotal role during liver inflammation, triggering fibrogenesis. The present paper discusses the potential role of markers in cell death and the inflammatory cascade leading to the severity of liver damage. We aim to present the clinical parameters and laboratory data in a cohort of 88 HCV-infected non-cirrhotic and 25 HCV cirrhotic patients, to determine the characteristic light microscopic (LM) and transmission electron microscopic (TEM) changes in their liver biopsies and to present the link between the severity of liver damage and the serum levels of cytokines and caspases. A matched HCV non-infected cohort was used for the comparison of serum inflammatory markers. We compared the inflammation in HCV individuals with a control group of 280 healthy individuals. We correlated the changes in inflammatory markers in different stages of the disease and the histology. We concluded that the serum levels of cytokine, chemokine, and cleaved caspase markers reveal the inflammatory status in HCV. Based upon the information provided by the changes in biomarkers the clinician can monitor the severity of HCV-induced liver damage. New oral well-tolerated treatment regimens for chronic hepatitis C patients can achieve cure rates of over 90%. Therefore, using the noninvasive biomarkers to monitor the evolution of the liver damage is an effective personalized medicine procedure to establish the severity of liver injury and its repair.
Topics: Apoptosis; Biomarkers; Case-Control Studies; Cell Death; Cytokines; Disease Susceptibility; Hepacivirus; Hepatitis C; Humans; Inflammation Mediators; Liver
PubMed: 34889885
DOI: 10.3390/cimb43030139 -
Bioscience Reports May 2019Human breast cancer is a malignant form of tumor with a relatively high mortality rate. Although esophageal cancer-related gene 4 (ECRG4) is thought to be a possible...
Human breast cancer is a malignant form of tumor with a relatively high mortality rate. Although esophageal cancer-related gene 4 (ECRG4) is thought to be a possible potent tumor suppressor gene that acts to suppress breast cancer, its precise role in this disease is not understood. Herein, we assess the correlation between ECRG4 expression and DNA methylation, probing the potential epigenetic regulation of ECRG4 in breast cancer. We analyzed ECRG4 promoter methylation via methylation-specific PCR (MSPCR), bisulfite sequencing, and a promoter reporter assay in human breast cancer cell lines and samples. Gene expression was assessed by quantitative real-time PCR (qPCR), while protein levels were assessed by Western blotting. CCK8 assays were used to quantify cell growth; Esophageal cancer-related gene 4 wound healing assays were used to assess cellular migration, while flow cytometry was used to assess apoptosis and cell cycle progression. Apoptosome formation was validated via CO-IP and Western blotting. We found that human breast cancer samples exhibited increased methylation of the ECRG4 promoter and decreased ECRG4 expression. Remarkably, the down-regulation of ECRG4 was highly associated with promoter methylation, and its expression could be re-activated via 5-aza-2'-deoxycytidine treatment to induce demethylation. ECRG4 overexpression impaired breast cancer cell proliferation and migration, and led to G0/G1 cell cycle phase arrest. Moreover, ECRG4 induced the formation of the Cytc/Apaf-1/caspase-9 apoptosome and promoted breast cancer cell apoptosis. ECRG4 is silenced in human breast cancer cells and cell lines, likely owing to promoter hypermethylation. ECRG4 may act as a tumor suppressor, inhibiting proliferation and migration, inducing G0/G1 phase arrest and apoptosis via the mitochondrial apoptotic pathway.
Topics: Apoptosis; Apoptosomes; Apoptotic Protease-Activating Factor 1; Base Sequence; Breast Neoplasms; Caspase 9; Cell Line, Tumor; Cell Movement; Cell Proliferation; DNA Methylation; Decitabine; Epigenesis, Genetic; Female; G1 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; Humans; Mitochondria; Promoter Regions, Genetic; RNA, Small Interfering; Signal Transduction; Tumor Suppressor Proteins
PubMed: 30918105
DOI: 10.1042/BSR20190087 -
International Journal of Cancer Sep 2015The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous,...
The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome-activating protein and investigate its potential pro-apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two-hybrid screen and further studied by deletion mutants, glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock-down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GISTs and histogenetically related cells including interstitial cells of Cajal (ICCs), "fibroblast-like cells" (FLCs) and ICC stem cells (ICC-SCs) was investigated by Northern blotting, reverse transcription-polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC-SCs stably transduced to re-express FAM96A was studied by xeno- and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC-SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC-SCs. Re-expression of FAM96A in GIST cells and transformed ICC-SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro-apoptotic tumor suppressor that is lost during GIST tumorigenesis.
Topics: Animals; Apoptosis; Apoptotic Protease-Activating Factor 1; Carrier Proteins; Cell Line; Cell Line, Tumor; Cell Transformation, Neoplastic; Gastrointestinal Stromal Tumors; Gene Expression; HEK293 Cells; Humans; Interstitial Cells of Cajal; Metalloproteins; Mice; Mice, Inbred NOD; Mice, Nude; Mice, SCID; Mitochondria; Tumor Suppressor Proteins; Zebrafish
PubMed: 25716227
DOI: 10.1002/ijc.29498