Activation Of Procaspase-3 Research Articles

Targeting type I DED interactions at the DED filament serves as a sensitive switch for cell fate decisions

Activation of procaspase-8 in the death effector domain (DED) filaments of the death-inducing signaling complex (DISC) is a key step in apoptosis. In this study, a rationally designed cell-penetrating peptide, DEDid, was engineered to mimic the h2b helical region of procaspase-8-DED2 containing a highly conservative FL motif. Furthermore, mutations were introduced into the DEDid binding site of the procaspase-8 type I interface. Additionally, our data suggest that DEDid targets other type I DED interactions such as those of FADD. Both approaches of blocking type I DED interactions inhibited CD95L-induced DISC assembly, caspase activation and apoptosis. We showed that inhibition of procaspase-8 type I interactions by mutations not only diminished procaspase-8 recruitment to the DISC but also destabilizedthe FADD core of DED filaments. Taken together, this study offers insights to develop strategies to target DED proteins, which may be considered in diseases associated with cell death and inflammation.

Caspase-8 promotes scramblase-mediated phosphatidylserine exposure and fusion of osteoclast precursors

Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss.Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.

A Non-Apoptotic Pattern of Caspase-9/Caspase-3 Activation During Differentiation of Human Embryonic Stem Cells into Cardiomyocytes.

In vitro studies have demonstrated that the differentiation of embryonic stem cells (ESCs) into cardiomyocytes requires activation of caspases through the mitochondrial pathway. These studies have relied on synthetic substrates for activity measurements, which can be misleading due to potential none-specific hydrolysis of these substrates by proteases other than caspases. Hence, caspase-9 and caspase-3 activation are investigated during the differentiation of human ESCs (hESCs) by directly assessing caspase-9 and -3 cleavage. Western blot reveals the presence of the cleaved caspase-9 prior to and during the differentiation of human ESCs (hESCs) into cardiomyocytes at early stages, which diminishes as the differentiation progresses, without cleavage and activation of endogenous procaspase-3. Activation of exogenous procaspase-3 by endogenous caspase-9 and subsequent cleavage of chromogenic caspase-3 substrate i.e. DEVD-pNA during the course of differentiation confirmes that endogenous caspase-9 has the potency to recognize and activate procaspase-3, but for reasons that are unknown to us fails to do so. These observations suggest the existence of distinct mechanisms of caspase regulation in differentiation as compared to apoptosis. Bioinformatics analysis suggests the presence of caspase-9 regulators, which may influence proteolytic function under specific conditions.

Arginine methylation of caspase-8 controls life/death decisions in extrinsic apoptotic networks

Procaspase-8 is a key mediator of death receptor (DR)-mediated pathways. Recently, the role of post-translational modifications (PTMs) of procaspase-8 in controlling cell death has received increasing attention. Here, using mass spectrometry screening, pharmacological inhibition and biochemical assays, we show that procaspase-8 can be targeted by the PRMT5/RIOK1/WD45 methylosome complex. Furthermore, two potential methylation sites of PRMT5 on procaspase-8, R233 and R435, were identified in silico. R233 and R435 are highly conserved in mammals and their point mutations are among the most common mutations of caspase-8 in cancer. The introduction of mutations at these positions resulted in inhibitory effects on CD95L-induced caspase-8 activity, effector caspase activation and apoptosis. In addition, we show that procaspase-8 can undergo symmetric di-methylation. Finally, the pharmacological inhibition of PRMT5 resulted in the inhibitory effects on caspase activity and apoptotic cell death. Taken together, we have unraveled the additional control checkpoint in procaspase-8 activation and the arginine methylation network in the extrinsic apoptosis pathway.

Deciphering DED assembly mechanisms in FADD-procaspase-8-cFLIP complexes regulating apoptosis

Fas-associated protein with death domain (FADD), procaspase-8, and cellular FLICE-inhibitory proteins (cFLIP) assemble through death-effector domains (DEDs), directing death receptor signaling towards cell survival or apoptosis. Understanding their three-dimensional regulatory mechanism has been limited by the absence of atomic coordinates for their ternary DED complex. By employing X-ray crystallography and cryogenic electron microscopy (cryo-EM), we present the atomic coordinates of human FADD-procaspase-8-cFLIP complexes, revealing structural insights into these critical interactions. These structures illustrate how FADD and cFLIP orchestrate the assembly of caspase-8-containing complexes and offer mechanistic explanations for their role in promoting or inhibiting apoptotic and necroptotic signaling. A helical procaspase-8-cFLIP hetero-double layer in the complex appears to promote limited caspase-8 activation for cell survival. Our structure-guided mutagenesis supports the role of the triple-FADD complex in caspase-8 activation and in regulating receptor-interacting protein kinase 1 (RIPK1). These results propose a unified mechanism for DED assembly and procaspase-8 activation in the regulation of apoptotic and necroptotic signaling across various cellular pathways involved in development, innate immunity, and disease.

Chitosan-encapsulated naringenin promotes ROS mediated through the activation of executioner caspase-3.

We previously reported that chitosan nanoparticle-encapsulated Naringenin (CS-NPs/NAR) could scavenge free radicals at lower doses and be cytotoxic to cancer cells. The current study continues to focus on the mechanism behind CS-NPs/NAR-induced breast cancer cell (MDA-MB-231) death. MDA-MB-231 cells were treated with higher concentrations (100, 200, and 200µg) of Chitosan nanoparticles (CS-NPs), naringenin (NAR), and chitosan-encapsulated naringenin (CS-NPs/NAR). The cell viability, proliferation, and oxidative stress parameters, such as nitric oxide [NO], xanthine oxidase (XOD), and xanthine dehydrogenase (XDH) levels, were analyzed. ROS levels were determined through DCFDA analysis. MTT-based cell cytotoxicity and BrdU cell proliferation analysis depicted the cytotoxicity effects (37% and 29% for 24 and 48h) and exhibited a reduction in the proliferation of MDA-MB-231 by CS-NPs/NAR. A significant increase in NO content, XOD, a decrease in XDH, and an increase in ROS levels were observed upon treatment with CS-NPs/NAR. Fluorescent images suggested the increase in the ROS level upon treatment with CS-NPs/NAR in cancer cells, and the results suggested that it could induce apoptosis. Further, to confirm this, the activity of caspase-3 was analyzed through western blotting, and the result suggested that the higher concentration of CS-NPs/NAR has increased the activation of procaspase3 when compared to free NAR. Hence, the current investigation concludes that high doses of CS-NPs/NAR induce and increase oxidative stress and so increased activation of procaspase3 may lead to cancer cell apoptosis and reduction in cell proliferation.

Molecular mechanism of Enteroaggregative Escherichia coli induced apoptosis in cultured human intestinal epithelial cells.

Background. Enteroaggregative Escherichia coli (EAEC) is an evolving etiological agent of acute and persistent diarrhoea worldwide. The previous study from our laboratory has reported the apoptosis-inducing activity of EAEC in human small intestinal and colonic epithelial cell lines. In the present investigation, we have explored the underlying mechanism of EAEC-induced apoptosis in human intestinal epithelial cell lines.Methods. INT-407 and HCT-15 cells were infected with EAEC-T8 and EAEC-pT8 (plasmid cured strain of EAEC-T8) separately. Cells cultured in the absence of bacteria served as a negative control in all the experiments. For the subsequent experiments, the molecular mechanism(s) of epithelial cell aposptosis was measured in EAEC infecting both the cell lines by flow cytometry, real-time PCR and Western blotting.Results and conclusions. EAEC was found to activate the intrinsic/mitochondrial apoptotic pathway in both the cell lines through upregulation of pro-apoptotic Bax and Bak, un-alteration/reduction in the level of anti-apoptotic Bcl-2 and Bcl-XL, decrease in mitochondrial transmembrane potential, accumulation of cytosolic cytochrome c leading to activation of procaspase-9 and procaspase-3, which ultimately resulted in DNA fragmentation and apoptosis. Further, an increased expression of Fas, activation of procaspase-8 and upregulation of pro-apoptotic Bid in the EAEC-infected cells indicated the involvement of extrinsic apoptotic pathway too in this process. Our finding has undoubtedly led to an increased understanding of EAEC pathogenesis, which may be helpful to develop an improved strategy to combat the infection.

Activation and Denitrosylation of Procaspase-3 in KA-induced Excitotoxicity.

It has been reported that activation of glutamate kainate receptor subunit 2 (GluK2) subunit-containing glutamate receptors and the following Fas ligand(FasL) up-regulation, caspase-3 activation, subsequently results in delayed apoptosis-like neuronal death in hippocampus CA1 subfield after cerebral ischemia. Nitric oxide-mediated S-nitrosylation might inhibit the procaspase activation, whereas denitrosylation might contribute to cleavage and activation of procaspases. The study aimed to elucidate the molecular mechanisms underlying procaspase-3 denitrosylation and activation following kainic acid (KA)-induced excitotoxicity in rat hippocampus. S-nitrosylation of procaspase-3 was detected by biotin-switch method. Activation of procaspase-3 was shown as cleavage of procaspase-3 detected by immunoblotting. FasL expression was detected by immunoblotting. Cresyl violets and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining were used to detect apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. KA led to the activation of procaspase-3 in a dose- and time-dependent manner, and the activation was inhibited by KA receptor antagonist NS102. Procaspase-3 was denitrosylated at 3 h after kainic acid administration, and the denitrosylation was reversed by SNP and GSNO. FasL ASODNs inhibited the procaspase-3 denitrosylation and activation. Moreover, thioredoxin reductase (TrxR) inhibitor auranofin prevented the denitrosylation and activation of procaspase-3 in rat hippocampal CA1 and CA3 subfields. NS102, FasL AS-ODNs, and auranofin reversed the KAinduced apoptosis and cell death in hippocampal CA1 and CA3 subfields. KA led to denitrosylation and activation of procaspase-3 via FasL and TrxR. Inhibition of procaspase-3 denitrosylation by auranofin, SNP, and GSNO played protective effects against KA-induced apoptosis-like neuronal death in rat hippocampal CA1 and CA3 subfields. These investigations revealed that the procaspase-3 undergoes an initial denitrosylation process before becoming activated, providing valuable insights into the underlying mechanisms and possible treatment of excitotoxicity.

The outer membrane protein Tp92 of Treponema pallidum delays human neutrophil apoptosis via the ERK, PI3K/Akt, and NF-κB pathways.

Syphilis is a persistent sexually transmitted disease caused by infiltration of the elusive pathogen Treponema pallidum. Despite the prevalence of human polymorphonuclear neutrophils (hPMNs) within cutaneous lesions, which are characteristic of incipient syphilis, their role in T. pallidum infection remains unclear. Tp92 is the only T. pallidum helical outer membrane protein that exhibits structural features similar to those of outer membrane proteins in other gram-negative bacteria. However, the functional mechanism of this protein in immune cells remains unclear. Neutrophils are short-lived cells that undergo innate apoptosis in response to external stimuli that typically influence this process. In this study, we determined that Tp92 impedes the activation of procaspase-3 via the ERK MAPK, PI3K/Akt, and NF-κB signaling pathways, consequently suppressing caspase-3 activity within hPMNs, and thereby preventing hPMNs apoptosis. Furthermore, Tp92 could also modulate hPMNs apoptosis by enhancing the expression of the anti-apoptotic protein Mcl-1, stimulating IL-8 secretion, and preserving the mitochondrial membrane potential. These findings provide valuable insights into the molecular mechanisms underlying T. pallidum infection and suggest potential therapeutic targets for syphilis treatment.

A gut-restricted glutamate carboxypeptidase II inhibitor reduces monocytic inflammation and improves preclinical colitis.

There is an urgent need to develop therapeutics for inflammatory bowel disease (IBD) because up to 40% of patients with moderate-to-severe IBD are not adequately controlled with existing drugs. Glutamate carboxypeptidase II (GCPII) has emerged as a promising therapeutic target. This enzyme is minimally expressed in normal ileum and colon, but it is markedly up-regulated in biopsies from patients with IBD and preclinical colitis models. Here, we generated a class of GCPII inhibitors designed to be gut-restricted for oral administration, and we interrogated efficacy and mechanism using in vitro and in vivo models. The lead inhibitor, (S)-IBD3540, was potent (half maximal inhibitory concentration = 4 nanomolar), selective, gut-restricted (AUCcolon/plasma > 50 in mice with colitis), and efficacious in acute and chronic rodent colitis models. In dextran sulfate sodium-induced colitis, oral (S)-IBD3540 inhibited >75% of colon GCPII activity, dose-dependently improved gross and histologic disease, and markedly attenuated monocytic inflammation. In spontaneous colitis in interleukin-10 (IL-10) knockout mice, once-daily oral (S)-IBD3540 initiated after disease onset improved disease, normalized colon histology, and attenuated inflammation as evidenced by reduced fecal lipocalin 2 and colon pro-inflammatory cytokines/chemokines, including tumor necrosis factor-α and IL-17. Using primary human colon epithelial air-liquid interface monolayers to interrogate the mechanism, we further found that (S)-IBD3540 protected against submersion-induced oxidative stress injury by decreasing barrier permeability, normalizing tight junction protein expression, and reducing procaspase-3 activation. Together, this work demonstrated that local inhibition of dysregulated gastrointestinal GCPII using the gut-restricted, orally active, small-molecule (S)-IBD3540 is a promising approach for IBD treatment.

Role of GPER in the expression and secretion of pro-inflammatory cytokines and calcium signaling in bone marrow-derived macrophages

Macrophages play an important role in mediating inflammation in numerous disease states by secreting pro-inflammatory cytokines including IL-1β and IL-18. Macrophages are driven into the M1-proinflammatory phenotype upon detection of pathogen-associated molecular patterns (PAMPs), or damage-associated molecular patterns (DAMPs). As macrophages are polarized into the M1 phenotype, they upregulate the expression of pro-inflammatory cytokines and components of the NLRP3 inflammasome. An additional activating stimulus is required to induce the assembly of the NLRP3 inflammasome, which facilitates the activation of pro-caspase 1 and the subsequent conversion of the pro-forms of both IL-18 and IL-1β into their mature forms for their secretion through gasdermin D membrane pores. A canonical activator of the NLRP3 inflammasome is extracellular ATP. ATP activation of the P2X7 receptor induces potassium efflux and calcium influx, both of which are necessary for NLRP3 inflammasome activation. Previous studies suggest that the G protein-coupled receptor (GPER) agonist G-1 reduced inflammation in animal disease models. However, the GPER-dependent effect of G-1 on pro-inflammatory cytokine expression and NLRP3 inflammasome activation in macrophages is unknown. In the current study, we utilized bone marrow-derived macrophages (BMDMs) from either WT or GPER-/- mice to test the hypothesis that G-1 reduces inflammatory cytokine production and NLRP3 inflammasome expression in a GPER-dependent manner. We found that the treatment of WT BMDMs with 100 nM G-1 for 24 hrs inhibited LPS-induced NLRP3, pro-IL-1β, IL-6, CCL5 and expression, and IL-1β secretion. Importantly, G-1 had no effect on GPER-/- BMDMs. To begin to determine if GPER modulates NLRP3 inflammasome activation, we characterized ATP-induced Ca2+ signals in both WT and GPER BMDMs with and without LPS pretreatment. No differences were observed between unprimed WT and GPER-/- BMDMs with respect to basal Ca2+ level, ATP-induced intracellular Ca2+ release, and subsequent bulk Ca2+ signal upon reestablishment of extracellular Ca2+. However, priming with LPS significantly increased ATP-induced intracellular Ca2+ release in both WT and GPER-/- BMDMs. Compared to WT cells, LPS-primed GPER-/- BMDMs exhibit significantly higher basal Ca2+ yet significantly lower ATP-induced Ca2+ release. Consistently, 15-minute treatment with 100 nM G-1 agonist strongly reduces ATP-induced Ca2+ release in WT BMDMs but had no effect in GPER-/- BMDMs. These results suggest a role for GPER in mediating Ca2+ uptake into IP3-sensitive intracellular compartments in BMDMs during LPS priming. In keeping with the known role for mobilization of intracellular Ca2+ in ATP-induced secretion of pro-inflammatory cytokines, preliminary data suggests that short-term G-1 pretreatment reduced IL-1β secretion induced by ATP in LPS-primed BMDMs. Overall, these data indicate a role for GPER in the priming and activation of the NLRP3 inflammasome. This research was supported by Des Moines University IOER Research & Grant Award #03-22-10. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

3D-QSAR assisted design, synthesis and pharmacological evaluation of novel substituted benzamides as procaspase-3 activators and anticancer agents

Novel substituted benzamides were designed using 3D-QSAR analysis, synthesized and screened using in vitro procaspase-3 kinase activation and MTT assays. For the rational design of the novel compounds, we performed 3D-QSAR study and generated statistically validated CoMFA (q2 = 0.736, r2cv = 0.729) and CoMSIA (q2 = 0.751, r2cv = 0.716) models. Generated contour maps using CoMFA and CoMSIA analysis suggested the requirement of steric bulkiness (aromatic ring) along with small electron-withdrawing groups (-Cl, -diCl, -F) for the design of novel compounds. CoMSIA, HBA, and HBD contour maps also suggested the requirement of functional groups (amide and hydrazide) for hydrogen bonding interactions. A total of 40 novel substituted benzamides were designed, and generated 3D-QSAR models were used for prediction of their activity prior to synthesis. ADMET prediction study was also carried out for the selection of better compounds with no toxicity and better physicochemical parameters for the synthesis. Finally, we synthesised and characterised a total of 10 substituted benzamide derivatives. Synthesized compounds were screened for their anticancer activities, in which compound 14c showed IC50 value of 3.13 µM and 6.01 µM against HT-29 and MDA-MB-231, respectively, compound 7c exhibited excellent anticancer activity with an IC50 value of 3.76 µM and 5/63 µM against HT-29 and MDA-MB-231, respectively. Furthermore, a procaspase-3 kinase activation assay was performed, in which both these compounds (7c, EC50 = 3.12 µM, 12b, EC50 = 2.93 µM and 14C EC50 =2.93) demonstrated activation of enzymatic potency and revealed that the anticancer activity of these compounds may be due to activation of the procaspase-3 enzyme. In vitro screening assays confirmed and validated the computational design of novel benzamide derivatives. Compounds 14c and 7c are our lead compounds for further development as anticancer agents based on the induction of apoptosis through procaspase-3 activation.

Transcriptional and Clonal Characterization of Cytotoxic T Cells in Crescentic Glomerulonephritis.

T-cell infiltration is a hallmark of crescentic GN (cGN), often caused by ANCA-associated vasculitis. Pathogenic T-cell subsets, their clonality, and downstream effector mechanisms leading to kidney injury remain to be fully elucidated. Single-cell RNA sequencing and T-cell receptor sequencing revealed activated, clonally expanded cytotoxic CD4 + and CD8 + T cells in kidneys from patients with ANCA-associated cGN. In experimental cGN, kidney-infiltrating CD8 + T cells expressed the cytotoxic molecule, granzyme B (GzmB), which induced apoptosis in renal tissue cells by activation of procaspase-3, and aggravated disease pathology. These findings describe a pathogenic function of (clonally expanded) cytotoxic T cells in cGN and identify GzmB as a mediator and potential therapeutic target in immune-mediated kidney disease. Crescentic GN (cGN) is an aggressive form of immune-mediated kidney disease that is an important cause of end stage renal failure. Antineutrophilic cytoplasmic antibody (ANCA)-associated vasculitis is a common cause. T cells infiltrate the kidney in cGN, but their precise role in autoimmunity is not known. Combined single-cell RNA sequencing and single-cell T-cell receptor sequencing were conducted on CD3 + T cells isolated from renal biopsies and blood of patients with ANCA-associated cGN and from kidneys of mice with experimental cGN. Functional and histopathological analyses were performed with Cd8a-/- and GzmB-/- mice. Single-cell analyses identified activated, clonally expanded CD8 + and CD4 + T cells with a cytotoxic gene expression profile in the kidneys of patients with ANCA-associated cGN. Clonally expanded CD8 + T cells expressed the cytotoxic molecule, granzyme B (GzmB), in the mouse model of cGN. Deficiency of CD8 + T cells or GzmB ameliorated the course of cGN. CD8 + T cells promoted macrophage infiltration and GzmB activated procaspase-3 in renal tissue cells, thereby increasing kidney injury. Clonally expanded cytotoxic T cells have a pathogenic function in immune-mediated kidney disease.

Acute radiofrequency electromagnetic radiation exposure impairs neurogenesis and causes neuronal DNA damage in the young rat brain

A mobile phone is now a commonly used device for digital media and communication among all age groups. Young adolescents use it for longer durations, which exposes them to radiofrequency electromagnetic radiation (RF-EMR). This exposure can lead to neuropsychiatric changes. The underlying cellular mechanism behind these changes requires detailed investigation. In the present study, we investigated the effect of RF-EMR emitted from mobile phones on young adolescent rat brains. Wistar rats (5 weeks, male) were exposed to RF-EMR signal (2115 MHz) at a head average specific absorption rate (SAR) of 1.51 W/kg continuously for 8 h. Higher level of lipid peroxidation, carbon-centered lipid radicals, and single-strand DNA damage was observed in the brain of rat exposed to RF-EMR. The number of BrdU-positive cells in the dentate gyrus (DG) decreased in RF-EMR-exposed rats, indicating reduced neurogenesis. RF-EMR exposure also induced degenerative changes and neuronal loss in DG neurons but had no effect on the CA3 and CA1 neurons of the hippocampus and cerebral cortex. The activity of Pro-caspase3 did not increase upon exposure in any of the brain regions, pointing out that degeneration observed in the DG region is not dependent on caspase activation. Results indicate that short-term acute exposure to RF-EMR induced the generation of carbon-centered lipid radicals and nuclear DNA damage, both of which likely played a role in the impaired neurogenesis and neuronal degeneration seen in the young brain's hippocampus region. The understanding of RF-EMR-induced alteration in the brain at the cellular level will help develop appropriate interventions for reducing its adverse impact.

Evaluation of central-metal effect on anticancer activity and mechanism of action of isostructural Cu(II) and Ni(II) complexes containing pyridine-2,6-dicarboxylate

Two Cu(II) (C1) and Ni(II) (C2) complexes were designed through the one-pot reaction of pyridine-2,6-dicarboxylic acid and 2-aminobenzimidazole respectively with copper(II) nitrate hexahydrate and nickel(II) nitrate hexahydrate. Both complexes were characterized by single-crystal X-ray diffraction and the distorted octahedral geometry was recognized for them. The MTT assay indicated that the complexes have a significant antiproliferative effect on BEL-7404 cells. IC50 values confirmed that C1 (IC50 = 0.56 μM) is several times more potent than C2 (IC50 = 5.13 μM). The similar cellular uptake of the complexes in mentioned cells led to this proposal that the production of ROS with different values can be the main reason for different cytotoxicity of the complexes. In this study, C1 and C2 caused BEL-7404 cells arrest at the G2/M and S phases, respectively. The expression of p53, Bax up-regulation, and Bcl-2 down-regulation and also activation of procaspase-9, and 3 indicated that apoptosis through a caspase-dependent mitochondrion pathway is a remarkable pathway in BEL-7404 cells treated by C1 while mechanistic studies proved that C2 induce death of BEL-7404 cells through the activation of RAGE/PI3KC3/Beclin 1 autophagic cell signaling pathway, more specifically. The cytostatic effect of the complexes in the BEL-7404 3D spheroid model was depicted.

Expression, purification, and characterization of c-FLIP tandem death effector domains from Escherichia coli

Cellular FLICE-like inhibitory protein (c-FLIP) regulates extrinsic apoptosis by controlling procaspase-8 activation through its tandem N-terminal death effector domains (DEDs). Here, we present the expression and purification of c-FLIP tandem DEDs (tDED) from Escherichia coli. We observed that the c-FLIPtDED maintains monomeric form under near-physiological pH condition in vitro. Our results also reveal a significant correlation between the pH conditions and the structure of c-FLIPtDED (F114A). The described methods and results would be helpful for follow-up study on the structural and functional of c-FLIP.

Design, Synthesis and Evaluation of Novel (E)-N'-((1-(4-chlorobenzyl)-1H-indol-3-yl)methylene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazides as Antitumor Agents.

Herein, we have designed and synthesized a series of the novel (E)-N'-((1-(4-chlorobenzyl)- 1H-indol-3-yl)methylene)-2-(4-oxoquinazolin-3(4H)-yl)acetohydrazides (5) as potent small molecules activating procaspase- 3. The compounds were designed by the amalgamation of structural features of PAC-1 (the first procaspase-3 activator) and oncrasin-1, one potential anticancer agent. The target acetohydrazides (5a-m) were prepared via the Niementowski condensation of anthranilic acid (1a) or 5-substituted-2-aminobenzoic acid (1b-m) and formamide. The compound libraries were evaluated for their cytotoxicity, caspase-3 activation, cell cycle analysis, and apoptosis. In addition, computational chemistry is also performed. A biological evaluation revealed that all thirteen compounds designed and synthesized showed strong cytotoxicity against three human cancer cell lines (SW620, colon cancer; PC-3, prostate cancer; NCI-H23, lung cancer) with eight compounds (5a, 5c-i, 5k), which were clearly more potent than both PAC-1 and oncrasin-1. In this series, four compounds, including 5c, 5e, 5f, and 5h, were the most potent members with approximately 4- to 5-fold stronger than the reference compounds PAC-1 and oncrasin-1 in terms of IC50. In comparison to 5-FU, these compounds were even 18- to 29-fold more potent in terms of cytotoxicity in three human cell lines tested. In the caspase activation assay, the caspase activity was activated to 285% by compound 5e compared to PAC-1, the first procaspase activating compound, which was used as a control. Our docking simulation revealed that compound 5e was a potent allosteric inhibitor of procaspase-3 through chelation of inhibitory zinc ion. Physicochemical and ADMET calculations for 5e provided useful information of its suitable absorption profile and some toxicological effects that need further optimization to be developed as a promising anticancer agent. Compound 5e has emerged as a potential hit for further design and development of caspases activators and anticancer agents.

Solution structure of c-FLIP death effector domains

The formation of death-inducing signaling complex (DISC) and death effector domain (DED) filament initiates extrinsic apoptosis. Recruitment and activation of procaspase-8 at the DISC are regulated by c-FLIP. The interaction between c-FLIP and procaspase-8 is mediated by their tandem DEDs (tDED). However, the structure of c-FLIPtDED and how c-FLIP interferes with procaspase-8 activation at the DISC remain elusive. Here, we solved the monomeric structure of c-FLIPtDED (F114G) at near physiological pH by solution nuclear magnetic resonance (NMR). Structural superimposition reveals c-FLIPtDED (F114G) adopts a structural topology similar to that of procaspase-8tDED. Our results provide a structural basis for understanding how c-FLIP interacts with procaspase-8 and the molecular mechanisms of c-FLIP in regulating cell death.

An engineered construct of cFLIP provides insight into DED1 structure and interactions

Cellular FLICE-like inhibitory protein (cFLIP) is a member of the Death Domain superfamily with pivotal roles in many cellular processes and disease states, including cancer and autoimmune disorders. In the context of the death-inducing signaling complex (DISC), cFLIP isoforms regulate extrinsic apoptosis by controlling procaspase-8 activation. The function of cFLIP is mediated through a series of protein-protein interactions, engaging the two N-terminal death effector domains (DEDs). Here, we solve the structure of an engineered DED1 domain of cFLIP using solution nuclear magnetic resonance (NMR) and we define the interaction with FADD and calmodulin, protein-protein interactions that regulate the function of cFLIP in the DISC. cFLIP DED1 assumes a canonical DED fold characterized by six α helices and is able to bind calmodulin and FADD through two separate interfaces. Our results clearly demonstrate the role of DED1 in the cFLIP/FADD association and contribute to the understanding of the assembly of DISC filaments.

Caspase-9 Activation of Procaspase-3 but Not Procaspase-6 Is Based on the Local Context of Cleavage Site Motifs and on Sequence.

Studying the interactions between a protease and its protein substrates at a molecular level is crucial for identifying the factors facilitating selection of particular proteolytic substrates and not others. These selection criteria include both the sequence and the local context of the substrate cleavage site where the active site of the protease initially binds and then performs proteolytic cleavage. Caspase-9, an initiator of the intrinsic apoptotic pathway, mediates activation of executioner procaspase-3 by cleavage of the intersubunit linker (ISL) at site 172IETD↓S. Although procaspase-6, another executioner, possesses two ISL cleavage sites (site 1, 176DVVD↓N; site 2, 190TEVD↓A), neither is directly cut by caspase-9. Thus, caspase-9 directly activates procaspase-3 but not procaspase-6. To elucidate this selectivity of caspase-9, we engineered constructs of procaspase-3 (e.g., swapping the ISL site, 172IETD↓S, with DVVDN and TEVDA) and procaspase-6 (e.g., swapping site 1, 176DVVD↓N, and site 2, 190TEVD↓A, with IETDS). Using the substrate digestion data of these constructs, we show here that the P4-P1' sequence of procaspase-6 ISL site 1 (DVVDN) can be accessed but not cleaved by caspase-9. We also found that caspase-9 can recognize the P4-P1' sequence of procaspase-6 ISL site 2 (TEVDA); however, the local context of this cleavage site is the critical factor that prevents proteolytic cleavage. Overall, our data have demonstrated that both the sequence and the local context of the ISL cleavage sites play a vital role in preventing the activation of procaspase-6 directly by caspase-9.