Kelvin Cain

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

cIAPs Block Ripoptosome Formation, a RIP1/Caspase-8 Containing Intracellular Cell Death Complex Differentially Regulated by cFLIP Isoforms

Summary The intracellular regulation of cell death pathways by cIAPs has been enigmatic. Here we show that loss of cIAPs promotes the spontaneous formation of an intracellular platform that activates either apoptosis or necroptosis. This 2 MDa intracellular complex that we designate “Ripoptosome” is necessary but not sufficient for cell death. It contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms. cFLIPL prevents Ripoptosome formation, whereas, intriguingly, cFLIPS promotes Ripoptosome assembly. When cIAPs are absent, caspase activity is the “rheostat” that is controlled by cFLIP isoforms in the Ripoptosome and decides if cell death occurs by RIP3-dependent necroptosis or caspase-dependent apoptosis. RIP1 is the core component of the complex. As exemplified by our studies for TLR3 activation, our data argue that the Ripoptosome critically influences the outcome of membrane-bound receptor triggering. The differential quality of cell death mediated by the Ripoptosome may cause important pathophysiological consequences during inflammatory responses.

The Apaf-1 apoptosome: a large caspase-activating complex

It is increasingly recognized that many key biological processes, including apoptosis, are carried out within very large multi-protein complexes. Apoptosis can be initiated by activation of death receptors or perturbation of the mitochondria causing the release of apoptogenic proteins, which result in the activation of caspases which are responsible for most of the biochemical and morphological changes observed during apoptosis. Caspases are normally inactive and require proteolytic processing for activity and this is achieved by the formation of large protein complexes known as the DISC (death inducing signalling complex) and the apoptosome. In the case of the latter complex, the central scaffold protein is a mammalian CED-4 homologue known as Apaf-1. This is an approximately 130 kDa protein, which in the presence of cytochrome c and dATP oligomerizes to form a very large (approximately 700-1400 kDa) apoptosome complex. The apoptosome recruits and processes caspase-9 to form a holoenzyme complex, which in turn recruits and activates the effector caspases. The apoptosome has been described in cells undergoing apoptosis, in dATP activated cell lysates and in reconstitution studies with recombinant proteins. Recent studies show that formation and function of the apoptosome can be regulated by a variety of factors including intracellular levels of K(+), inhibitor of apoptosis proteins (IAPs), heat shock proteins and Smac/Diablo. These various factors thus ensure that the apoptosome complex is only fully assembled and functional when the cell is irrevocably destined to die.

Caspase Activation Involves the Formation of the Aposome, a Large (∼700 kDa) Caspase-activating Complex

In mammals, apoptotic protease-activating factor 1 (Apaf-1), cytochrome c, and dATP activate caspase-9, which initiates the postmitochondrial-mediated caspase cascade by proteolytic cleavage/activation of effector caspases to form active ∼60-kDa heterotetramers. We now demonstrate that activation of caspases either in apoptotic cells or following dATP activation of cell lysates results in the formation of two large but different sized protein complexes, the “aposome” and the “microaposome”. Surprisingly, most of the DEVDase activity in the lysate was present in the aposome and microaposome complexes with only small amounts of active caspase-3 present as its free ∼60-kDa heterotetramer. The larger aposome complex (M r = ∼ 700,000) contained Apaf-1 and processed caspase-9, -3, and -7. The smaller microaposome complex (M r = ∼ 200,000–300,000) contained active caspase-3 and -7 but little if any Apaf-1 or active caspase-9. Lysates isolated from control THP.1 cells, prior to caspase activation, showed striking differences in the distribution of key apoptotic proteins. Apaf-1 and procaspase-7 may be functionally complexed as they eluted as an ∼200–300-kDa complex, which did not have caspase cleavage (DEVDase) activity. Procaspase-3 and -9 were present as separate and smaller 60–90-kDa (dimer) complexes. During caspase activation, Apaf-1, caspase-9, and the effector caspases redistributed and formed the aposome. This resulted in the processing of the effector caspases, which were then released, possibly bound to other proteins, to form the microaposome complex.

A Death Effector Domain Chain DISC Model Reveals a Crucial Role for Caspase-8 Chain Assembly in Mediating Apoptotic Cell Death

Summary Formation of the death-inducing signaling complex (DISC) is a critical step in death receptor-mediated apoptosis, yet the mechanisms underlying assembly of this key multiprotein complex remain unclear. Using quantitative mass spectrometry, we have delineated the stoichiometry of the native TRAIL DISC. While current models suggest that core DISC components are present at a ratio of 1:1, our data indicate that FADD is substoichiometric relative to TRAIL-Rs or DED-only proteins; strikingly, there is up to 9-fold more caspase-8 than FADD in the DISC. Using structural modeling, we propose an alternative DISC model in which procaspase-8 molecules interact sequentially, via their DED domains, to form a caspase-activating chain. Mutating key interacting residues in procaspase-8 DED2 abrogates DED chain formation in cells and disrupts TRAIL/CD95 DISC-mediated procaspase-8 activation in a functional DISC reconstitution model. This provides direct experimental evidence for a DISC model in which DED chain assembly drives caspase-8 dimerization/activation, thereby triggering cell death.

Reconstitution of the Death-Inducing Signaling Complex Reveals a Substrate Switch that Determines CD95-Mediated Death or Survival

The death-inducing signaling complex (DISC) is critical for initiation of death-receptor-mediated apoptosis; however, paradoxically, CD95 also signals for cell survival. Here, we reconstitute a functional DISC using only purified CD95, FADD, and procaspase-8 and unveil a two-step activation mechanism involving both dimerization and proteolytic cleavage of procaspase-8 that is obligatory for death-receptor-induced apoptosis. Initially, dimerization yields active procaspase-8 with a very restricted substrate repertoire, limited to itself or c-FLIP. Proteolytic cleavage is then required to fully activate caspase-8, thereby permitting DISC-mediated cleavage of the critical exogenous apoptotic substrates, caspase-3 and Bid. This switch in catalytic activity and substrate range is a key determinant of DISC signaling, as cellular expression of noncleavable procaspase-8 mutants, which undergo DISC-mediated oligomerization, but not cleavage, fails to initiate CD95-induced apoptosis. Thus, using the reconstituted DISC, we have delineated a crucial two-step activation mechanism whereby activated death receptor complexes can trigger death or survival.

HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen species

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

Protein Profiling of Plasma Membranes Defines Aberrant Signaling Pathways in Mantle Cell Lymphoma

We used shotgun proteomics to identify plasma membrane and lipid raft proteins purified from B cells obtained from mantle cell lymphoma (MCL) patients in leukemic phase. Bioinformatics identified 111 transmembrane proteins, some of which were profiled in primary MCL cases, MCL-derived cell lines, and normal B cells using RT-PCR and Western blotting. Several transmembrane proteins, including CD27, CD70, and CD31 (PECAM-1), were overexpressed when compared with normal B cells. CD70 was up-regulated (>10-fold) in three of five MCL patients along with its cognate receptor CD27, which was up-regulated (4–9-fold) in five of five patients, suggesting that MCL cells may undergo autocrine stimulation via this signaling pathway. Activated calpain I and protein kinase C βII were also detected in the plasma membranes, suggesting that these proteins are constitutively active in MCL. Protein kinase C βII has been associated with lipid rafts, and shotgun proteomics/protein profiling revealed that key lipid raft proteins, raftlin (four of five patients) and CSK (C-terminal Src kinase)-binding protein (Cbp)/phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG) (four of four patients) were down-regulated in MCL. Levels of other known lipid raft proteins, such as Lyn kinase and flotillin 1, were similar to normal B cells. However, 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, was associated with lipid rafts and was up-regulated ∼7-fold in MCL compared with normal B cells. Significantly inhibitors of 5-LO activity (AA861) and 5-LO-activating protein (FLAP) (MK886, its activating enzyme) induced apoptosis in MCL cell lines and primary chronic lymphocytic leukemia cells, indicating an important role for the leukotriene biosynthetic pathway in MCL and other B cell malignancies. Thus, using shotgun proteomics and mRNA and protein expression profiling we identified a subset of known and unknown transmembrane proteins with aberrant expression in MCL plasma membranes. These proteins may play a role in the pathology of the disease and are potential therapeutic targets in MCL.

Metabolic effects of TiO2 nanoparticles, a common component of sunscreens and cosmetics, on human keratinocytes

The long-term health risks of nanoparticles remain poorly understood, which is a serious concern given their prevalence in the environment from increased industrial and domestic use. The extent to which such compounds contribute to cellular toxicity is unclear, and although it is known that induction of oxidative stress pathways is associated with this process, the proteins and the metabolic pathways involved with nanoparticle-mediated oxidative stress and toxicity are largely unknown. To investigate this problem further, the effect of TiO2 on the HaCaT human keratinocyte cell line was examined. The data show that although TiO2 does not affect cell cycle phase distribution, nor cell death, these nanoparticles have a considerable and rapid effect on mitochondrial function. Metabolic analysis was performed to identify 268 metabolites of the specific pathways involved and 85 biochemical metabolites were found to be significantly altered, many of which are known to be associated with the cellular stress response. Importantly, the uptake of nanoparticles into the cultured cells was restricted to phagosomes, TiO2 nanoparticles did not enter into the nucleus or any other cytoplasmic organelle. No other morphological changes were detected after 24-h exposure consistent with a specific role of mitochondria in this response.

Caspases-3 and -7 are activated in goniothalamin-induced apoptosis in human Jurkat T-cells

Goniothalamin, a plant styrylpyrone derivative isolated from Goniothalamus andersonii, induced apoptosis in Jurkat T‐cells as assessed by the externalisation of phosphatidylserine. Immunoblotting showed processing of caspases‐3 and ‐7 with the appearance of their catalytically active large subunits of 17 and 19 kDa, respectively. Activation of these caspases was further evidenced by detection of poly(ADP‐ribose) polymerase cleavage (PARP). Pre‐treatment with the caspase inhibitor benzyloxycarbonyl‐Val‐Ala‐Asp fluoromethyl ketone (Z‐VAD.FMK) blocked apoptosis and the resultant cleavage of these caspases and PARP. Our results demonstrate that activation of at least two effector caspases is a key feature of goniothalamin‐induced apoptosis in Jurkat T‐cells.