Apo cytochrome c inhibits caspases by preventing apoptosome formation

doi:10.1016/j.bbrc.2004.05.084

Biochemical and Biophysical Research Communications

Volume 319, Issue 3, 2 July 2004, Pages 944-950

https://doi.org/10.1016/j.bbrc.2004.05.084 Get rights and content

Abstract

Caspases are cysteine proteases and potent inducers of apoptosis. Their activation and activity is therefore tightly regulated. There are several mechanisms by which caspases can be activated but one key pathway involves release of holo cytochrome c from mitochondria into the cytoplasm. Cytoplasmic holo cytochrome c binds to apoptotic protease activating factor-1 (Apaf-1), driving the formation of an Apaf-1 oligomer (the apoptosome) which in turn binds and activates caspase-9. Previously we showed that the apo form of cytochrome c (lacking heme) can bind Apaf-1 and block both holo-dependent caspase activation in cell extracts and Bax-induced apoptosis in cells. Here we tested the ability of apo cytochrome c to inhibit caspase-9 activation induced by recombinant Apaf-1. Furthermore, using purified proteins and size exclusion chromatography we show that apo cytochrome c prevents holo cytochrome c-dependent apoptosome formation.

Section snippets

Materials and methods

Recombinant cytochrome c and Apaf-1. Human apo cytochrome c and recombinant Apaf-1 were expressed and purified as described [21], [22]. Briefly, His-tagged cytochrome c was expressed in bacteria and purified on Nickel agarose and S-Sepharose. His tagged Apaf-1 was expressed in Sf2 cells and also purified on Nickel agarose. Holo cytochrome c (equine) was purchased from Sigma.

Apaf-1pull-down assay. An Apaf-1 enriched fraction from 293 cells derived S-100 extracts were obtained as described in [22]

Results

The holo-form of cytochrome c (containing heme) is a small globular protein that can trigger Apaf-1 dependent caspase activation by binding to Apaf-1. In contrast, the apo form lacking heme cannot activate caspases and we assumed that the apo form could not bind Apaf-1. Therefore, it was unexpected that both the holo- and apo-forms of cytochrome c interacted with Apaf-1 in 293 cell extracts [21] (Fig. 1A). Our previous study described the binding of apo cytochrome c to Apaf-1 and inhibition of

Discussion

Cytochrome c is a nuclear gene and after translation apo cytochrome c translocates into mitochondria where heme is added to form the holo protein. The holo protein remains in the mitochondria, functioning in electron transport until it is released into the cytosol by apoptotic stimuli at which point it can bind Apaf-1, triggering caspase activation. Several studies have identified amino acid residues, predominantly lysines, in cytochrome c that are necessary for caspase activation [26], [27].

Acknowledgements

We thank Nancy Martin and Linda Miller for technical support. Also we thank Dr. I. Daar and Dr. P. Kaldis (LPDS, NCI-Frederick) for helpful discussion and critical reading of the manuscript.

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For instance, PARC (also known as CUL9) was found to be the ubiquitin ligase responsible for the ubiquitination and proteasomal degradation of cytochrome c [42]. Interestingly, the cytochrome c without the heme group (apo-cytochrome c) inhibits apoptosome formation by directly binding to Apaf-1 and preventing Apaf-1 oligomerization [43]. Intracellular Leucine-Rich alpha-2-Glycoprotein-1 (LRG1) is another protein that competes with Apaf-1 for cytochrome c binding in MCF-7 breast cancer cells preventing Apaf-1 heptamerization and apoptosome formation.
Deregulation of apoptosis is associated with various pathologies, such as neurodegenerative disorders at one end of the spectrum and cancer at the other end. Generally speaking, differentiated cells like cardiomyocytes, skeletal myocytes and neurons exhibit low levels of Apaf-1 (Apoptotic protease activating factor 1) protein suggesting that down-regulation of Apaf-1 is an important event contributing to the resistance of these cells to apoptosis. Nonetheless, upregulation of Apaf-1 has not emerged as a common phenomenon in pathologies associated with enhanced neuronal cell death, i.e., neurodegenerative diseases. In cancer, on the other hand, Apaf-1 downregulation is a common phenomenon, which occurs through various mechanisms including mRNA hyper-methylation, gene methylation, Apaf-1 localization in lipid rafts, inhibition by microRNAs, phosphorylation, and interaction with specific inhibitors. Due to the diversity of these mechanisms and involvement of other factors, defining the exact contribution of Apaf-1 to the development of cancer in general and neurodegenerative disorders, in particular, is complicated. The current review is an attempt to provide a comprehensive image of Apaf-1's contribution to the pathologies observed in cancer and neurodegenerative diseases with the emphasis on the therapeutic aspects of Apaf-1 as an important target in these pathologies.
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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.
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Wild-type and mutant Cyt c constructs were verified by sequencing. To prepare recombinant Cyt c proteins, BL21 bacteria were transformed with Cyt c plasmids together with pET-heme lyase plasmid (which encodes the Cyt c heme lyase) in order to express the heme-containing Cyt c proteins [67]. Positive clones were grown in LB medium and protein expression was induced by addition of 0.3 mM isopropyl-1-thio-D-galactopyranoside (IPTG, Invitrogen) at 25 °C for 16 h. Bacterial cells were collected by centrifugation and resuspended in native lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), supplemented with protease inhibitors leupeptin, chymostatin, antipain, and pepstatin A (all 2 μg/ml) containing lysozyme and incubated on ice for 30 min and then lysed by sonication.
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.
Apoptosome formation upon overexpression of native and truncated Apaf-1 in cell-free and cell-based systems
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For cloning of Apaf-1 we used pcDNA3.1 vector. Apaf-1 sequence was PCR amplified using high fidelity PrimeSTAR GXL DNA Polymerase (Clontech) from FastBAC vector containing the His tagged Apaf-1 [21]. PCR-amplified N-terminal (1–416) and C-terminal (395–550) luciferase fragments were derived from pGL3 plasmid encoding Photinus pyralis luciferase and used for constructing split reporter [22,23].
Apaf-1 is a cytosolic multi-domain protein in the apoptosis regulatory network. When cytochrome c releases from mitochondria; it binds to WD-40 repeats of Apaf-1 molecule and induces oligomerization of Apaf-1. Here in, a split luciferase assay was used to compare apoptosome formation in cell-free and cell-based systems. This assay uses Apaf-1 tagged with either N-terminal fragment or C-terminal fragment of P. pyralis luciferase. In cell based-system, the apoptosome formation is induced inside the cells which express Apaf-1 tagged with complementary fragments of luciferase while in cell-free system, the apoptosome formation is induced in extracts of the cells. In cell-free system, cytochrome c dependent luciferase activity was observed with full length Apaf-1. However, luciferase activity due to apoptosome formation was much higher in cell based system compared to cell-free system. The truncated Apaf-1 which lacks WD-40 repeats (ΔApaf-1) interacted with endogenous Apaf-1 in a different fashion compared to native form as confirmed by different retention time of eluate in gel filtration and binding to affinity column. The interactions between endogenous Apaf-1 and ΔApaf-1 is stronger than its interaction with native exogenous Apaf-1 as indicated by dominant negative effect of ΔApaf-1 on caspase-3 processing.
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In spite of various reports, the mechanistic aspect of regulation of apoptosome formation and caspase activation in in-vivo/in-vitro systems triggered by cytochrome c is not clearly understood. Importantly, apocytochrome c (i.e., freshly synthesized cytochrome c) that does not contain heme-moiety also interacts with Apaf-1 and inhibits apoptosome assembly (Martin et al., 2004). These studies clearly suggest that apocytochrome c functions as prosurvival molecule, while holocytochrome c serves as key survival molecules as a part of electron transport chain.
Cancer cells are resistant to conventional chemotherapy and radiotherapy, however, the molecular mechanisms of resistance to therapy remain unclear. Cellular survival machinery protects mitochondrial integrity against endogenous or exogenous stresses. Prodeath molecules orchestrate around mitochondria to initiate and execute cell death in cancer, and also play an underappreciated role in survival of cancer cells. Prosurvival mechanisms can operate at mitochondrial and postmitochondrial levels to attenuate core apoptotic death program. It is intriguing to explore how prosurvival and prodeath molecules crosstalk to regulate mitochondrial functions leading to increased cancer cell survival. This review describes some putative survival mechanisms at mitochondria, which may play a role in designing effective agents for cancer prevention and therapy. These survival pathways may also have significance in understanding other human pathophysiological conditions including diabetes, cardiovascular, autoimmune, and neurodegenerative diseases.
Binding conformation prediction between human acetylcholinesterase and cytochrome c using molecular modeling methods
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The acetylcholinesterase (AChE) is important to terminate acetylcholine-mediated neurotransmission at cholinergic synapses. The pivotal role of AChE in apoptosome formation through the interactions with cytochrome c (Cyt c) was demonstrated in recent study. In order to investigate the proper binding conformation between the human AChE (hAChE) and human Cyt c (hCyt c), macro-molecular docking simulation was performed using DOT 2.0 program. The hCyt c was bound to peripheral anionic site (PAS) on hAChE and binding mode of the docked conformation was very similar to the reported crystal structure of the AChE and fasciculin-II (Fas-II) complex. Two 10 ns molecular dynamics (MD) simulations were carried out to refine the binding mode of docked structure and to observe the differences of the binding conformations between the absent (Apo) and presence (Holo) of heme group. The key hydrogen bonding residues between hAChE and hCyt c proteins were found in Apo and Holo systems, as well as each Tyr341 and Trp286 residue of hAChE was participated in cation-pi (π) interactions with Lys79 of hCyt c in Apo and Holo systems, respectively. From the present study, although the final structures of the Apo and Holo systems have similar binding pattern, several differences were investigated in flexibilities, interface interactions, and interface accessible surface areas. Based on these results, we were able to predict the reasonable binding conformation which is indispensable for apoptosome formation.

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^☆: Abbreviations: Apaf-1, apoptotic protease activating factor-1; CARD, caspase recruitment domain; DEVD-AFC, N-acetyl Asp-Glu-Val-Asp-7-amino-4-fluoromethyl coumarin.

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Apo cytochrome c inhibits caspases by preventing apoptosome formation☆

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

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