Apoptosome Assembly Research Articles

Конформаційні зміни флуоресцентно міченого G-актину при взаємодії з цитохромом c як свідчення можливого залучення білків цитоскелета до процесу апоптозу

Existing data suggest the involvement of cytoskeletal proteins, particularly actin, in the process of apoptosis. However, the exact role of actin remains unclear. The aim of this study was to investigate the potential functional interaction of this protein with one of the key participants in apoptosis, cytochrome c. We utilized fluorescent labeling of amino acid residues of rat brain-derived b/g-actin in its monomeric form (G-actin) using the fluorescent dye 3-(4-carboxybenzoyl) quinoline2-carboxaldehyde (CBQCA) to monitor induced molecular changes. It is well known that CBQCA reacts with primary amines in the presence of cyanide to form a fluorescent product. However, we also discovered the formation of another fluorescent derivative when the dye reacted with the phenolic group of free tyrosine, as well as its residues in peptides and proteins, independent of cyanide presence. During the investigation of conformational changes in labeled G-actin molecules, we found that the fluorescence intensity varied in the spectral region characteristic of the reaction product with tyrosine residues, but not with amine groups. Specifically, cytochrome c at micromolar concentrations decreased fluorescence in a dose-dependent manner, indicating direct interaction with G-actin. Actin-specific toxins and other factors affecting its polymerization also reduced the fluorescence intensity of labeled actin, without significantly influencing the effect of cytochrome c. At the same time, cytochrome c had no effect on the fluorescence of inactivated labeled actin. Thus, by utilizing the previously undescribed CBQCA labeling of tyrosine residues, we were able to study conformational changes in the actin molecule and establish the interaction of this protein with cytochrome c. It can be hypothesized that actin is involved in apoptosis as a regulator of apoptosome assembly, particularly through its interaction with cytochrome c.

Retraction: A Short Caspase-3 Isoform Inhibits Chemotherapy-Induced Apoptosis by Blocking Apoptosome Assembly

Retraction: A Short Caspase-3 Isoform Inhibits Chemotherapy-Induced Apoptosis by Blocking Apoptosome Assembly

Changes in the Acetylcholinesterase Enzymatic Activity in Tumor Development and Progression.

Acetylcholinesterase is a well-known protein because of the relevance of its enzymatic activity in the hydrolysis of acetylcholine in nerve transmission. In addition to the catalytic action, it exerts non-catalytic functions; one is associated with apoptosis, in which acetylcholinesterase could significantly impact the survival and aggressiveness observed in cancer. The participation of AChE as part of the apoptosome could explain the role in tumors, since a lower AChE content would increase cell survival due to poor apoptosome assembly. Likewise, the high Ach content caused by the reduction in enzymatic activity could induce cell survival mediated by the overactivation of acetylcholine receptors (AChR) that activate anti-apoptotic pathways. On the other hand, in tumors in which high enzymatic activity has been observed, AChE could be playing a different role in the aggressiveness of cancer; in this review, we propose that AChE could have a pro-inflammatory role, since the high enzyme content would cause a decrease in ACh, which has also been shown to have anti-inflammatory properties, as discussed in this review. In this review, we analyze the changes that the enzyme could display in different tumors and consider the different levels of regulation that the acetylcholinesterase undergoes in the control of epigenetic changes in the mRNA expression and changes in the enzymatic activity and its molecular forms. We focused on explaining the relationship between acetylcholinesterase expression and its activity in the biology of various tumors. We present up-to-date knowledge regarding this fascinating enzyme that is positioned as a remarkable target for cancer treatment.

Enhancing Apoptosome Assembly via Mito‐Biomimetic Lipid Nanocarrier for Cancer Therapy

AbstractApoptosis is the natural programmed cell death process, which is responsible for abnormal cell clearance. However, many cancer cells develop various mechanisms to escape apoptosis through interrupting apoptosome assembly, which is a key step to initiate apoptosis. This promotes tumorigenesis and drug resistance, and thus, poses a great challenge in cancer treatment. Herein, a biomimetic lipid nanocarrier mimicking mitochondrial Cytochrome C (Cyt C) binding is developed. Cardiolipin, the major phospholipid of mitochondrial inner membrane, is introduced as the main component in biomimetic liposomal formulation. With the help of cardiolipin, Cyt C is sufficiently loaded in liposome based on electrostatic and hydrophobic interaction with cardiolipin. Lonidamine (LND) is added in hydrophobic phase of liposome to modulate the metabolic activity within cancer cells and sensitize the cells to Cyt C‐induced apoptosis. The results suggest that LND reduces ATP level and creates favorable environment for Cyt C induced apoptosome assembly, exhibiting higher apoptosis level and anti‐tumor efficacy in vitro and in vivo. The conjugation of a tumor‐homing peptide, LinTT1, on the nanovesicle, increases the efficacy due to enhanced tumor accumulation. Overall, this biomimetic lipid nanocarrier proves to be an efficient delivery system with great potential of pro‐apoptosis cancer therapy.

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.

F-actin-rich territories coordinate apoptosome assembly and caspase activation during DNA damage-induced intrinsic apoptosis.

The actin cytoskeleton is a ubiquitous participant in cellular functions that maintain viability, but how it controls programmed cell death is not well understood. Here we show that in response to DNA damage, human cells form a juxtanuclear F-actin-rich territory that coordinates the organized progression of apoptosome assembly to caspase activation. This cytoskeletal compartment is created by the actin nucleation factors JMY, WHAMM, and the Arp2/3 complex, and it excludes proteins that inhibit JMY and WHAMM activity. Within the territory, mitochondria undergo outer membrane permeabilization and JMY localization overlaps with punctate structures containing the core apoptosome components cytochrome c and Apaf-1. The F-actin-rich area also encompasses initiator caspase-9 and clusters of a cleaved form of executioner caspase-3 but restricts accessibility of the caspase inhibitor XIAP. The clustering and potency of caspase-3 activation are positively regulated by the amount of actin polymerized by JMY and WHAMM. These results indicate that JMY-mediated actin reorganization functions in apoptotic signaling by coupling the biogenesis of apoptosomes to the localized processing of caspases. [Media: see text].

APAF1-Binding Long Noncoding RNA Promotes Tumor Growth and Multidrug Resistance in Gastric Cancer by Blocking Apoptosome Assembly.

Chemotherapeutics remain the first choice for advanced gastric cancers (GCs). However, drug resistance and unavoidable severe toxicity lead to chemotherapy failure and poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in tumor progression in many cancers, including GC. Here, through RNA screening, an apoptotic protease‐activating factor 1 (APAF1)‐binding lncRNA (ABL) that is significantly elevated in cancerous GC tissues and an independent prognostic factor for GC patients is identified. Moreover, ABL overexpression inhibits GC cell apoptosis and promotes GC cell survival and multidrug resistance in GC xenograft and organoid models. Mechanistically, ABL directly binds to the RNA‐binding protein IGF2BP1 via its KH1/2 domain, and then IGF2BP1 further recognizes the METTL3‐mediated m6A modification on ABL, which maintains ABL stability. In addition, ABL can bind to the WD1/WD2 domain of APAF1, which competitively prevent cytochrome c from interacting with APAF1, blocking apoptosome assembly and caspase‐9/3 activation; these events lead to resistance to cell death in GC cells. Intriguingly, targeting ABL using encapsulated liposomal siRNA can significantly enhance the sensitivity of GC cells to chemotherapy. Collectively, the results suggest that ABL can be a potential prognostic biomarker and therapeutic target in GC.

Diversity in the intrinsic apoptosis pathway of nematodes

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.

Capsaicin induces mitochondrial dysfunction and apoptosis in anaplastic thyroid carcinoma cells via TRPV1-mediated mitochondrial calcium overload

Anaplastic thyroid cancer (ATC) is a rare malignancy and has a poor prognosis due to its aggressive behavior and resistance to treatments. Calcium (Ca2+) serves as a ubiquitous cellular second messenger and influences several tumor behaviors. Therefore, Ca2+ modulation is expected to be a novel therapeutic target in cancers. However, whether Ca2+ 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 Ca2+ influx by TRPV1 activation, resulting in disequilibrium of intracellular calcium homeostasis. The rapidly increased cytosolic Ca2+ 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 Ca2+ 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.

Nwd1 Regulates Neuronal Differentiation and Migration through Purinosome Formation in the Developing Cerebral Cortex.

SummaryEngagement of neural stem/progenitor cells (NSPCs) into proper neuronal differentiation requires the spatiotemporally regulated generation of metabolites. Purines are essential building blocks for many signaling molecules. Enzymes that catalyze de novo purine synthesis are assembled as a huge multienzyme complex called “purinosome.” However, there is no evidence of the formation or physiological function of the purinosome in the brain. Here, we showed that a signal transduction ATPases with numerous domains (STAND) protein, NACHT and WD repeat domain-containing 1 (Nwd1), interacted with Paics, a purine-synthesizing enzyme, to regulate purinosome assembly in NSPCs. Altered Nwd1 expression affected purinosome formation and induced the mitotic exit and premature differentiation of NSPCs, repressing neuronal migration and periventricular heterotopia. Overexpression/knockdown of Paics or Fgams, other purinosome enzymes, in the developing brain resulted in a phenocopy of Nwd1 defects. These findings indicate that strict regulation of purinosome assembly/disassembly is crucial for maintaining NSPCs and corticogenesis.

Molecular insights on cytochrome c and nucleotide regulation of apoptosome function and its implication in cancer

Cytochrome c (Cyt c) released from mitochondria interacts with Apaf-1 to form the heptameric apoptosome, which initiates the caspase cascade to execute apoptosis. Although lysine residue at 72 (K72) of Cyt c plays an important role in the Cyt c-Apaf-1 interaction, the underlying mechanism of interaction between Cyt c and Apaf-1 is still not clearly defined. Here we identified multiple lysine residues including K72, which are also known to interact with ATP, to play a key role in Cyt c-Apaf-1 interaction. Mutation of these lysine residues abrogates the apoptosome formation causing inhibition of caspase activation. Using in-silico molecular docking, we have identified Cyt c-binding interface on Apaf-1. Although mutant Cyt c shows higher affinity for Apaf-1, the presence of Cyt c-WT restores the apoptosome activity. ATP addition modulates only mutant Cyt c binding to Apaf-1 but not WT Cyt c binding to Apaf-1. Using TCGA and cBioPortal, we identified multiple mutations in both Apaf-1 and Cyt c that are predicted to interfere with apoptosome assembly. We also demonstrate that transcript levels of various enzymes involved with dATP or ATP synthesis are increased in various cancers. Silencing of nucleotide metabolizing enzymes such as ribonucleotide reductase subunit M1 (RRM1) and ATP-producing glycolytic enzymes PKM2 attenuated ATP production and enhanced caspase activation. These findings suggest important role for lysine residues of Cyt c and nucleotides in the regulation of apoptosome-dependent apoptotic cell death as well as demonstrate how these mutations and nucleotides may have a pivotal role in human diseases such as cancer.

Does Oxidation of Mitochondrial Cardiolipin Trigger a Chain of Antiapoptotic Reactions?

Oxidative stress causes selective oxidation of cardiolipin (CL), a four-tail lipid specific for the inner mitochondrial membrane. Interaction with oxidized CL transforms cytochrome c into peroxidase capable of oxidizing even more CL molecules. Ultimately, this chain of events leads to the pore formation in the outer mitochondrial membrane and release of mitochondrial proteins, including cytochrome c, into the cytoplasm. In the cytoplasm, cytochrome c promotes apoptosome assembly that triggers apoptosis (programmed cell death). Because of this amplification cascade, even an occasional oxidation of a single CL molecule by endogenously formed reactive oxygen species (ROS) might cause cell death, unless the same CL oxidation triggers a separate chain of antiapoptotic reactions that would prevent the CL-mediated apoptotic cascade. Here, we argue that the key function of CL in mitochondria and other coupling membranes is to prevent proton leak along the interface of interacting membrane proteins. Therefore, CL oxidation should increase proton permeability through the CL-rich clusters of membrane proteins (CL islands) and cause a drop in the mitochondrial membrane potential (MMP). On one hand, the MMP drop should hinder ROS generation and further CL oxidation in the entire mitochondrion. On the other hand, it is known to cause rapid fission of the mitochondrial network and formation of many small mitochondria, only some of which would contain oxidized CL islands. The fission of mitochondrial network would hinder apoptosome formation by preventing cytochrome c release from healthy mitochondria, so that slowly working protein quality control mechanisms would have enough time to eliminate mitochondria with the oxidized CL. Because of these two oppositely directed regulatory pathways, both triggered by CL oxidation, the fate of the cell appears to be determined by the balance between the CL-mediated proapoptotic and antiapoptotic reactions. Since this balance depends on the extent of CL oxidation, mitochondria-targeted antioxidants might be able to ensure cell survival in many pathologies by preventing CL oxidation.

Cross Talk Between Cellular Redox State and the Antiapoptotic Protein Bcl-2

B cell lymphoma-2 (Bcl-2) was discovered over three decades ago and is the prototype antiapoptotic member of the Bcl-2 family that comprises proteins with contrasting effects on cell fate. First identified as a consequence of chromosomal translocation (t 14:18) in human lymphoma, subsequent studies have revealed mutations and/or gene copy number alterations as well as post-translational modifications of Bcl-2 in a variety of human cancers. The canonical function of Bcl-2 is linked to its ability to inhibit mitochondrial membrane permeabilization, thereby regulating apoptosome assembly and activation by blocking the cytosolic translocation of death amplification factors. Of note, the identification of specific domains within the Bcl-2 family of proteins (Bcl-2 homology domains; BH domains) has not only provided a mechanistic insight into the various interactions between the member proteins but has also been the impetus behind the design and development of small molecule inhibitors and BH3 mimetics for clinical use. Recent Advances: Aside from its role in maintaining mitochondrial integrity, recent evidence provides testimony to a novel facet in the biology of Bcl-2 that involves an intricate cross talk with cellular redox state. Bcl-2 overexpression modulates mitochondrial redox metabolism to create a "pro-oxidant" milieu, conducive for cell survival. However, under states of oxidative stress, overexpression of Bcl-2 functions as a redox sink to prevent excessive buildup of reactive oxygen species, thereby inhibiting execution signals. Emerging evidence indicates various redox-dependent transcriptional changes and post-translational modifications with different functional outcomes. Understanding the complex interplay between Bcl-2 and the cellular redox milieu from the standpoint of cell fate signaling remains vital for a better understanding of pathological states associated with altered redox metabolism and/or aberrant Bcl-2 expression. Based on its canonical functions, Bcl-2 has emerged as a potential druggable target. Small molecule inhibitors of Bcl-2 and/or other family members with similar function, as well as BH3 mimetics, are showing promise in the clinic. The emerging evidence for the noncanonical activity linked to cellular redox metabolism provides a novel avenue for the design and development of diagnostic and therapeutic strategies against cancers refractory to conventional chemotherapy by the overexpression of this prosurvival protein.

Optimal pathways for the assembly of the Apaf-1·cytochrome c complex into apoptosome.

The formation of a heptameric apoptosome is a crucial event in the intrinsic cell death pathway. Considerable progress has been made towards unraveling the constituents and the structure of the apoptosome as well as the mechanism of apoptosome-mediated caspase-9 activation. However, a significant gap remains in the understanding of this process, i.e., how seven Apaf-1·cytochrome c complexes stepwisely assemble into an apoptosome. Here, we construct a biophysical model that incorporates current biochemical knowledge about the formation of apoptosome. We propose 11 elementary routes and enumerate all 2047 possible assembly pathways from the Apaf-1·cytochrome c complex to the heptameric apoptosome. By combining mathematical analysis and numerical simulation, we find that two elementary routes are the most favorable biochemical reaction routes and there are 52 optimal assembly pathways which are economical and relatively fast. Our study yields the first comprehensive analysis of apoptosome assembly and provides insights into complex assembly pathways.

The DNA replication protein Cdc6 inhibits the microtubule-organizing activity of the centrosome

The centrosome serves as a major microtubule-organizing center (MTOC). The Cdc6 protein is a component of the pre-replicative complex and a licensing factor for the initiation of chromosome replication and localizes to centrosomes during the S and G2 phases of the cfell cycle of human cells. This cell cycle-dependent localization of Cdc6 to the centrosome motivated us to investigate whether Cdc6 negatively regulates MTOC activity and to determine the integral proteins that comprise the pericentriolar material (PCM). Time-lapse live-cell imaging of microtubule regrowth revealed that Cdc6 depletion increased microtubule nucleation at the centrosomes and that expression of Cdc6 in Cdc6-depleted cells reversed this effect. This increase and decrease in microtubule nucleation correlated with the centrosomal intensities of PCM proteins such as γ-tubulin, pericentrin, CDK5 regulatory subunit-associated protein 2 (CDK5RAP2), and centrosomal protein 192 (Cep192). The regulation of microtubule nucleation and the recruitment of PCM proteins to the centrosome required Cdc6 ATPase activity, as well as a centrosomal localization of Cdc6. These results suggest a novel function for Cdc6 in coordinating centrosome assembly and function.

The Cellular Apoptosis Susceptibility Protein (CAS) Promotes Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis and Cell Proliferation

A signature event during the cell intrinsic apoptotic pathway is mitochondrial outer membrane permeabilization, leading to formation of the apoptosome, a caspase activation complex. The cellular apoptosis susceptibility protein (CAS) can facilitate apoptosome assembly by stimulating nucleotide exchange on Apaf-1 following binding of cytochrome c. We report here that CAS expression itself is up-regulated during tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, and knockdown of CAS renders cells resistant to TRAIL. We find that TRAIL induces up-regulation of CAS in a posttranscriptional, caspase-8-dependent manner through degradation of cIAP1, an E3 ligase that targets CAS for ubiquitin-dependent proteasomal degradation. We identified a novel signaling pathway whereby caspase-8 engages a feedforward cascade that leads to CAS up-regulation and amplifies the apoptotic signal. Furthermore, in silico analysis revealed that expression of CAS is up-regulated at both the mRNA and DNA levels in human breast tumors, consistent with its role in promoting cell proliferation. Overexpression of various oncogenes led to CAS up-regulation in non-transformed cells. Intriguingly, oncogene-induced CAS up-regulation also resulted in greater susceptibility to TRAIL-induced cell death, consistent with its proapoptotic function. These findings suggest that CAS plays contrasting roles in proliferation and apoptosis and that overexpression of CAS in tumors could serve as a potential biomarker to guide therapeutic choices.

Modeling of interaction between cytochrome c and the WD domains of Apaf-1: bifurcated salt bridges underlying apoptosome assembly.

BackgroundBinding of cytochrome c, released from the damaged mitochondria, to the apoptotic protease activating factor 1 (Apaf-1) is a key event in the apoptotic signaling cascade. The binding triggers a major domain rearrangement in Apaf-1, which leads to oligomerization of Apaf-1/cytochrome c complexes into an apoptosome. Despite the availability of crystal structures of cytochrome c and Apaf-1 and cryo-electron microscopy models of the entire apoptosome, the binding mode of cytochrome c to Apaf-1, as well as the nature of the amino acid residues of Apaf-1 involved remain obscure.ResultsWe investigated the interaction between cytochrome c and Apaf-1 by combining several modeling approaches. We have applied protein-protein docking and energy minimization, evaluated the resulting models of the Apaf-1/cytochrome c complex, and carried out a further analysis by means of molecular dynamics simulations. We ended up with a single model structure where all the lysine residues of cytochrome c that are known as functionally-relevant were involved in forming salt bridges with acidic residues of Apaf-1. This model has revealed three distinctive bifurcated salt bridges, each involving a single lysine residue of cytochrome c and two neighboring acidic resides of Apaf-1. Salt bridge-forming amino acids of Apaf-1 showed a clear evolutionary pattern within Metazoa, with pairs of acidic residues of Apaf-1, involved in bifurcated salt bridges, reaching their highest numbers in the sequences of vertebrates, in which the cytochrome c-mediated mechanism of apoptosome formation seems to be typical.ConclusionsThe reported model of an Apaf-1/cytochrome c complex provides insights in the nature of protein-protein interactions which are hard to observe in crystallographic or electron microscopy studies. Bifurcated salt bridges can be expected to be stronger than simple salt bridges, and their formation might promote the conformational change of Apaf-1, leading to the formation of an apoptosome. Combination of structural and sequence analyses provides hints on the evolution of the cytochrome c-mediated apoptosis.ReviewersThis article was reviewed by Andrei L. Osterman, Narayanaswamy Srinivasan, Igor N. Berezovsky, and Gerrit Vriend (nominated by Martijn Huynen).Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0059-4) contains supplementary material, which is available to authorized users.

Characterization of phase I metabolism of resibufogenin and evaluation of the metabolic effects on its antitumor activity and toxicity.

Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has displayed great potential as a chemotherapeutic agent in oncology. However, it is a digoxin-like compound that also exhibits extremely cardiotoxic effects. The present study aimed to characterize the metabolic behaviors of RB in humans as well as to evaluate the metabolic effects on its bioactivity and toxicity. The phase I metabolic profile in human liver microsomes was characterized systemically, and the major metabolite was identified as marinobufagenin (5β-hydroxylresibufogenin, 5-HRB) by liquid chromatography-mass spectrometry and nuclear magnetic imaging techniques. Both cytochrome P450 (P450) reaction phenotyping and inhibition assays using P450-selective chemical inhibitors demonstrated that CYP3A4 was mainly involved in RB 5β-hydroxylation with much higher selectivity than CYP3A5. Kinetic characterization demonstrated that RB 5β-hydroxylation in both human liver microsomes and human recombinant CYP3A4 obeyed biphasic kinetics and displayed similar apparent kinetic parameters. Furthermore, 5-HRB could significantly induce cell growth inhibition and apoptosis in A549 and H1299 by facilitating apoptosome assembly and caspase activation. Meanwhile, 5-HRB displayed very weak cytotoxicity of human embryonic lung fibroblasts, and in mice there was a greater tolerance to acute toxicity. In summary, CYP3A4 dominantly mediated 5β-hydroxylation and was found to be a major metabolic pathway of RB in the human liver, whereas its major metabolite (5-HRB) displayed better druglikeness than its parent compound RB. Our findings lay a solid foundation for RB metabolism studies in humans and encourage further research on the bioactive metabolite of RB.

Amyloid β binds procaspase-9 to inhibit assembly of Apaf-1 apoptosome and intrinsic apoptosis pathway

Apoptosis is essential in the death process induced by Amyloid-β (Aβ), a major constituent of diffuse plaques found in Alzheimer's disease patients. However, we have found that caspase activation and cell death induced by staurosporine, employed to induce the intrinsic mitochondria-dependent apoptotic pathway, were significantly reduced by 42 amino-acid Aβ42, implying that the peptide also has a negative effect on the apoptotic process. The inhibitory effect of Aβ42 on the apoptotic pathway is associated with its interaction with procaspase-9 and consequent inhibition of Apaf-1 apoptosome assembly. We detected the inhibitory effect in the early stage (<8h) of apoptosis, but later caspase activation becomes obvious. Thus we inferred that the inhibitory process on apoptosis begins at an early stage, and the later robust activation surpasses it. We propose that the apoptotic manifestation in Aβ-treated cells is a combined consequence of those anti- and pro-apoptotic processes.

Changes in Apaf-1 Conformation That Drive Apoptosome Assembly

Apoptosome assembly is highly regulated in the intrinsic cell death pathway. To better understand this step, we created an improved model of the human apoptosome using a crystal structure of full length Apaf-1 and a single particle, electron density map at ~9.5 Å resolution. The apoptosome model includes N-terminal domains of Apaf-1, cognate β-propellers, and cytochrome c. A direct comparison of Apaf-1 in the apoptosome and as a monomer reveals conformational changes that occur during the first two steps of assembly. This includes an induced-fit mechanism for cytochrome c binding to regulatory β-propellers, which is dependent on shape and charge complementarity, and a large rotation of the nucleotide binding module during nucleotide exchange. These linked conformational changes create an extended Apaf-1 monomer and drive apoptosome assembly. Moreover, the N-terminal CARD in the inactive Apaf-1 monomer is not shielded from other proteins by β-propellers. Hence, the Apaf-1 CARD may be free to interact with a procaspase-9 CARD either before or during apoptosome assembly. Irrespective of the timing, the end product of assembly is a holo-apoptosome with an acentric CARD-CARD disk and tethered pc-9 catalytic domains. Subsequent activation of pc-9 leads to a proteolytic cascade and cell death.