PHAPI, CAS, and Hsp70 Promote Apoptosome Formation by Preventing Apaf-1 Aggregation and Enhancing Nucleotide Exchange on Apaf-1

Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390, USA.
Molecular cell (Impact Factor: 14.02). 05/2008; 30(2):239-47. DOI: 10.1016/j.molcel.2008.03.014
Source: PubMed


During apoptosis, cytochrome c is released from mitochondria to the cytosol, where it binds Apaf-1. The Apaf-1/cytochrome c complex then oligomerizes either into heptameric caspase-9-activating apoptosome, which subsequently activates caspase-3 and caspase-7, or bigger inactive aggregates, depending on the availability of nucleotide dATP/ATP. A tumor suppressor protein, PHAPI, enhances caspase-9 activation by promoting apoptosome formation through an unknown mechanism. We report here the identification of cellular apoptosis susceptibility protein (CAS) and heat shock protein 70 (Hsp70) as mediators of PHAPI activity. PHAPI, CAS, and Hsp70 function together to accelerate nucleotide exchange on Apaf-1 and prevent inactive Apaf-1/cytochrome c aggregation. CAS expression is induced by multiple apoptotic stimuli including UV irradiation. Knockdown of CAS by RNA interference (RNAi) in cells attenuates apoptosis induced by UV light and causes endogenous Apaf-1 to form aggregates. These studies indicated that PHAPI, CAS, and Hsp70 play an important regulatory role during apoptosis.

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Available from: Fenghe Du, Oct 08, 2015
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    • "The cellular actions of Hsp70 are mediated mainly by its physical association with a number of co-chaperones. For example, Hsp70 was reported to associate with APAF1, an important apoptosis regulator, to modulate apoptosome formation [10], [11], [12]. In binding to Hsp70, C terminus of Hsc70-interactiong protein (CHIP) and BAG-1 may serve as a link between the chaperone and the proteasome, perhaps aiding in the targeting of substrates for degradation [13], [14], [15]. "
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    ABSTRACT: The endotoxin-mediated production of pro-inflammatory cytokines plays an important role in the pathogenesis of liver disorders. Heat shock protein (Hsp70) overexpression has established functions in lipopolysaccharide (LPS)-mediated inflammatory response. However, little is known about the role of Hsp70 activity in LPS signaling. We hypothesized that inhibition of Hsp70 substrate binding activity can ameliorate LPS-induced liver injury by decreasing induction of pro-inflammatory factors. In this study, C57/BL6 mice were injected intraperitoneally with LPS and 2-phenylethynesulfonamide (PES), an inhibitor of Hsp70 substrate binding activity. We found that i. PES prevented LPS-induced increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, infiltration of inflammatory cells, and liver cell apoptosis; ii. PES reduced inducible nitric oxide synthase (iNOS) protein expression as well as serum nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) content in LPS-stimulated mice; iii. PES reduced the mRNA level of iNOS, TNF-α, and IL-6 in LPS-stimulated liver. iiii. PES attenuated the degradation of inhibitor of κB-α (IκB-α) as well as the phosphorylation and nuclear translocation of nuclear factor-κB (NF-κB) in LPS-stimulated liver. Similar changes in the protein expression of inflammatory markers, IκB-α degradation, and NF-κB phosphorylation and nuclear translocation were observed in RAW 264.7 cells. Further mechanistic studies revealed that PES remarkably reduced the elevation of [Ca(2+)]i and intracellular pH value (pHi) in LPS-stimulated RAW 264.7 cells. Furthermore, PES significantly reduced the increase in Na(+)/H(+) exchanger 1 (NHE1) association to Hsp70 in LPS-stimulated macrophages and liver, suggesting that NHE1-Hsp70 interaction is required for the involvement of NHE1 in the inflammation response. In conclusion, inhibition of Hsp70 substrate binding activity in vivo reduces the induction of pro-inflammatory factors and prevents LPS-induced liver injury likely by disrupting NHE1-Hsp70 interaction which consequently reduces the activation of IκB-α-NF-κB pathway in liver.
    PLoS ONE 06/2013; 8(6):e67582. DOI:10.1371/journal.pone.0067582 · 3.23 Impact Factor
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    • "In addition to the previously investigated alpha and beta tubulins, here we analyzed the effects of these two diatom diets on the expression levels of genes which are known to have a primary role in generic stress responses, defense systems (e.g. aldehyde, free fatty acid and free radical detoxification) or apoptosis regulation in other organisms, from humans to marine organisms [19], [20], [21], [22], [23], [24], [25], [26], [27] (Figure 1). We expected an activation of enzymes and proteins involved in stress responses (e.g heat shock proteins, phase I and phase II enzymes), but, in particular, we hypothesized expression level increases of enzymes that could detoxify and/or metabolize toxic diatom PUAs. "
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    ABSTRACT: Diatoms are dominant photosynthetic organisms in the world's oceans and are considered essential in the transfer of energy through marine food chains. However, these unicellular plants at times produce secondary metabolites such as polyunsaturated aldehydes and other products deriving from the oxidation of fatty acids that are collectively termed oxylipins. These cytotoxic compounds are responsible for growth inhibition and teratogenic activity, potentially sabotaging future generations of grazers by inducing poor recruitment in marine organisms such as crustacean copepods. Here we show that two days of feeding on a strong oxylipin-producing diatom (Skeletonema marinoi) is sufficient to inhibit a series of genes involved in aldehyde detoxification, apoptosis, cytoskeleton structure and stress response in the copepod Calanus helgolandicus. Of the 18 transcripts analyzed by RT-qPCR at least 50% were strongly down-regulated (aldehyde dehydrogenase 9, 8 and 6, cellular apoptosis susceptibility and inhibitor of apoptosis IAP proteins, heat shock protein 40, alpha- and beta-tubulins) compared to animals fed on a weak oxylipin-producing diet (Chaetoceros socialis) which showed no changes in gene expression profiles. Our results provide molecular evidence of the toxic effects of strong oxylipin-producing diatoms on grazers, showing that primary defense systems that should be activated to protect copepods against toxic algae can be inhibited. On the other hand other classical detoxification genes (glutathione S-transferase, superoxide dismutase, catalase, cytochrome P450) were not affected possibly due to short exposure times. Given the importance of diatom blooms in nutrient-rich aquatic environments these results offer a plausible explanation for the inefficient use of a potentially valuable food resource, the spring diatom bloom, by some copepod species.
    PLoS ONE 10/2011; 6(10):e26850. DOI:10.1371/journal.pone.0026850 · 3.23 Impact Factor
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    • "In this scenario, the apoptosome could bind hemiactivated caspase-3/pc-3 dimers at adjacent sites that are not sterically blocked by the disk or by bound pc-9 catalytic domains, without losing pc-9 activity. Since intracellular concentrations of pc-9 and pc-3 are approximately 10 and 20 nM, respectively (Kim et al., 2008; Stoka et al., 2001), the initiator apoptosome would activate pc-3 in an efficient manner. However, in some cells the pc-3 concentration has been estimated to be as high as approximately 100 nM (Yin et al., 2006). "
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    ABSTRACT: Activation of procaspase-9 on the apoptosome is a pivotal step in the intrinsic cell death pathway. We now provide further evidence that caspase recruitment domains of pc-9 and Apaf-1 form a CARD-CARD disk that is flexibly tethered to the apoptosome. In addition, a 3D reconstruction of the pc-9 apoptosome was calculated without symmetry restraints. In this structure, p20 and p10 catalytic domains of a single pc-9 interact with nucleotide binding domains of adjacent Apaf-1 subunits. Together, disk assembly and pc-9 binding create an asymmetric proteolysis machine. We also show that CARD-p20 and p20-p10 linkers play important roles in pc-9 activation. Based on the data, we propose a proximity-induced association model for pc-9 activation on the apoptosome. We also show that pc-9 and caspase-3 have overlapping binding sites on the central hub. These binding sites may play a role in pc-3 activation and could allow the formation of hybrid apoptosomes with pc-9 and caspase-3 proteolytic activities.
    Structure 08/2011; 19(8):1084-96. DOI:10.1016/j.str.2011.07.001 · 5.62 Impact Factor
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