The Pro-Apoptotic Proteins, Bid and Bax, Cause a Limited Permeabilization of the Mitochondrial Outer Membrane That Is Enhanced by Cytosol

Kluck, Ruth M.; Esposti, Mauro Dogli; Perkins, Guy; Renken, Christian; Kuwana, Tomomi; Bossy‐Wetzel, Ella; Goldberg, Martin W.; Allen, Terence D; Barber, Michael J.; Green, Douglas R.; Newmeyer, Donald D.

doi:10.1083/jcb.147.4.809

Cited by 304 publications

(239 citation statements)

References 87 publications

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“…This is contrary to the behaviour of mitochondria as ascertained by the distribution curve of reference enzyme cytochrome oxidase. Such a curve is markedly shifted towards the low-density zones of the gradient after aFas injection, probably as a result of the swelling of the mitochondria that takes place during apoptosis [16]. It is also worthwhile to mention that these results show that sedimentable lysosomal enzymes, present in the liver homogenates after aFas injection, are truly associated with lysosomes.…”

Section: Figure 2 Unsedimentable (A) and Total (B) Activities Of Sulfmentioning

confidence: 71%

“…Fractionation of the homogenate by differential centrifugation was achieved using the method of de Duve et al [20]. A nuclear fraction N, a heavy mitochondrial fraction M, a light mitochondrial fraction L, a microsomal fraction P and a soluble fraction S were successively isolated by integrated forces respectively of 10 000, 33 000, 250 000 and 3 000 000 g · min using the fractionation scheme described by these authors [16]. For isopycnic centrifugation, preformed linear density gradients of Percoll (27-90 %) with 0.25 M sucrose as a solvent were used and centrifugation was performed at 25 000 rev./min in a Beckman SW 65 rotor for 60 min.…”

Section: In Vivo Experimentsmentioning

confidence: 99%

“…Hepatocyte apoptosis was assessed by measuring the activity of the major executioner caspase, caspase 3, with a site-specific tetrapeptide substrate (DEVDase activity) and by determining the percentage of sulfite oxidase released in the high-speed supernatant of the homogenate. This enzyme is located in the intermembrane space of mitochondria [15] and is co-released with cytochrome c into the cytosol during apoptosis [16]. The measurement of this enzymatic activity with cytochrome c as electron acceptor (sulfite cytochrome c reductase) allows quantification of the proportion of mitochondria whose outer membrane is permeabilized.…”

Section: Introductionmentioning

confidence: 99%

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Lysosomes and Fas-mediated liver cell death

Wattiaux

Coninck

Thirion

et al. 2007

Biochemical Journal

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A number of studies, mostly performed ex vivo, suggest that lysosomes are involved in apoptosis as a result of a release of their cathepsins into the cytosol. These enzymes could then contribute to the permeabilization of the outer mitochondrial membrane; they could also activate effector caspases. The present study aims at testing whether the membrane of liver lysosomes is disrupted during Fas-mediated cell death of hepatocytes in vivo, a process implicated in several liver pathologies. Apoptosis was induced by injecting mice with aFas (anti-Fas antibody). The state of lysosomes was assessed by determining the proportion of lysosomal enzymes (β-galactosidase, β-glucuronidase, cathepsin C and cathepsin B) present in homogenate supernatants, devoid of intact lysosomes, and by analysing the behaviour in differential and isopycnic centrifugation of β-galactosidase. Apoptosis was monitored by measuring caspase 3 activity (DEVDase) and the release of sulfite cytochrome c reductase, an enzyme located in the mitochondrial intermembrane space. Results show that an injection of 10 µg of aFas causes a rapid and large increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase. This modifies neither the proportion of unsedimentable lysosomal enzyme in the homogenates nor the behaviour of lysosomes in centrifugation. Experiments performed with a lower dose of aFas (5 µg) indicate that unsedimentable lysosomal hydrolase activity increases in the homogenate after injection but with a marked delay with respect to the increase in DEVDase activity and in unsedimentable sulfite cytochrome c reductase.Comparative experiments ex vivo performed with Jurkat cells show an increase in unsedimentable lysosomal hydrolases, but much later than caspase 3 activation, and a release of dipeptidyl peptidase III and DEVDase into culture medium. It is proposed that the weakening of lysosomes observed after aFas treatment in vivo and ex vivo results from a necrotic process that takes place late after initiation of apoptosis.

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Section: Figure 2 Unsedimentable (A) and Total (B) Activities Of Sulfmentioning

confidence: 71%

Section: In Vivo Experimentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Lysosomes and Fas-mediated liver cell death

Wattiaux

Coninck

Thirion

et al. 2007

Biochemical Journal

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“…Despite all the data implicating the PT-pore in cell death, a number of caveats have been raised against the PT-pore: Some investigators failed to see a dependence on the PT-pore for Bax-and Bid-induced apoptosis [16][17][18][19]. Also, in some apoptosis scenarios Bax-induced release of cytochrome c could not be inhibited by bongkrekic acid and or CsA [20].…”

Section: Evidence Against the Pt-pore In Cell Death And Modificationsmentioning

confidence: 99%

The permeability transition pore in cell death

2007

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The permeability transition pore (PT-pore) is a multi-component protein aggregate in mitochondria that comprises factors in the inner as well as in the outer mitochondrial membrane. This complex has two functions: firstly, it regulates the integration of oxidative phosphorylation into the cellular energy household and secondly, it induces cell death when converted into an unspecific channel. The latter causes a collapse of the mitochondrial membrane potential and activates a chain of events that culminate in the demise of the cell. It has been controversial for some time whether the PT-pore is causative for or only amplifies a signal of cell death but novel results confirm a central role of this protein complex for cell death induction. While a considerable body of data exist on its subunit composition, recent genetic knock-out experiments suggest that the identity of the core factors of the PT-pore is still unresolved. Moreover, accumulating evidence point to a much more complex composition of this protein complex than anticipated. Here, we review the current knowledge of its subunit composition, the evidence of a role in cell death, and we propose a model for the activation of the PT-pore for cell death. The role of the PT-pore in apoptosisThe permeability transitionThe permeability transition (PT) is a sudden and sustained increase of the permeability of the inner mitochondrial membrane for solutes smaller than 1.5 kD. This has catastrophic consequences for this organelle. The mitochondrial membrane potential m , which relies on the im-permeability of the inner membrane for protons, breaks down and with it the ability of the cell to synthesize ATP. The ensuing blockade of the respiratory chain leads to the generation of reactive oxygen intermediates (ROIs), most likely via the direct transfer of electrons to molecular oxygen. Also, the high concentration of solutes in the mitochondrial matrix generates an osmotic pressure, which drives the influx of water molecules and the expansion of the extensively folded inner membrane and eventually disrupts the outer membrane leading to the release of factors that execute the suicide programme of apoptosis. Thus, the self-destruction of mitochondria via the PT is followed by the demise of the cell as a whole. This process of mitochondrial destruction was first observed in vitro but was dismissed at the time as an artefact because, among other reasons, it was inconceivable why mitochondria should dispose of a process to dismantle themselves. This changed, however, when it was shown that the PT is mediated by a protein complex, the so-called permeability transition pore (PT-pore), and that various physiological and pharmacological reagents can act on this pore and modify the deadly response of mitochondria. Finally, the realisation that cells can mount an active suicide response that is mediated in large part by mitochondria placed the PT-pore firmly on the map of researchers. In particular, the use of cyclosporine A (CsA) that can inhibit the PT-pore subunit cyc...

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“…12 Mitochondria are deeply involved in the regulation of apoptosis. 13,14 The family of Bcl-2-related proteins function like checkpoints through which survival and death signals must pass before they determine the cell fate. Indeed, a major site of activity of the Bcl-2 proteins is the mitochondrial membrane, promoting or preventing cytochrome c release.…”

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confidence: 99%

Irs–2 Mediates the Antiapoptotic Effect of Insulin in Neonatal Hepatocytes

et al. 2004

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To assess the role of insulin action and inaction in the liver, immortalized hepatocyte cell lines have been generated from insulin receptor substrate (IRS)-2 ؊/؊ and wild-type mice. Using this model, we have recently demonstrated that the lack of IRS-2 in neonatal hepatocytes resulted in insulin resistance. In the current study, we show that immortalized neonatal hepatocytes undergo apoptosis on serum withdrawal, with caspase-3 activation and DNA laddering occurring earlier in the absence of IRS-2. Insulin rescued wild-type hepatocytes from serum withdrawal-induced caspase-3 activation and DNA fragmentation in a dose-dependent manner, but it failed to rescue hepatocytes lacking IRS-2. In IRS-2 ؊/؊ cells, insulin failed to phosphorylate Bad. I n the liver, regulation of apoptosis is essential for development and hepatic homeostatic mechanisms. 1 Thus, liver hyperplasia during development or regeneration may be aided by inhibition of apoptosis. Conversely, liver atrophy occurs by apoptosis of liver cells in the absence of regeneration (for review, see Patel et al. 2 ). Progression of the apoptotic program can be inhibited at different levels depending on the origin of the death stimulus. In this regard, insulin promotes survival in a number of cell types. This effect is mediated by a complex network of intracellular signaling pathways. 3 The cascade begins when the activated insulin receptor (IR) phosphorylates several docking proteins, including the insulin receptor substrate (IRS) family 4 and Shc. Then, phosphorylated IRSs (IRS-1, -2, -3, and -4) connect with the phosphatidylinositol 3-kinase (PI 3-kinase) signaling pathway, 5 which plays a central role in the metabolic actions elicited by insulin, and the Ras/MAPK pathway, a major regulatory pathway for gene expression. 6 Among the members of the IRS family, IRS-2 triggers insulin actions in the liver. In fact, mice deficient in IRS-2 develop hepatic insulin resistance and fatty liver along with ␤-cell failure, 7-10 producing a severe type-2 diabetic phenotype. At the mo-

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The Pro-Apoptotic Proteins, Bid and Bax, Cause a Limited Permeabilization of the Mitochondrial Outer Membrane That Is Enhanced by Cytosol

Cited by 304 publications

References 87 publications

Lysosomes and Fas-mediated liver cell death

Lysosomes and Fas-mediated liver cell death

The permeability transition pore in cell death

Irs–2 Mediates the Antiapoptotic Effect of Insulin in Neonatal Hepatocytes

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