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Biomedical subjects

John D Robertson

Publications and source records attributed to John D Robertson.

8 recordsLinked to original sources
PubMed2004

Caspase-2 permeabilizes the outer mitochondrial membrane and disrupts the binding of cytochrome c to anionic phospholipids.

Caspases are cysteine proteases that play a central role in the execution of apoptosis. Recent evidence indicates that caspase-2 is activated early in response to genotoxic stress and can function as an upstream modulator of the mitochondrial apoptotic pathway. In particular, we have shown previously that fully processed caspase-2 can permeabilize the outer mitochondrial membrane and cause cytochrome c and Smac/DIABLO release from these organelles. Using permeabilized cells, isolated mitochondria, and protein-free liposomes, we now report that this effect is direct and depends neither on the presence or cleavage of other proteins nor on a specific phospholipid composition of the liposomal membrane. Interestingly, caspase-2 was also shown to disrupt the interaction of cytochrome c with anionic phospholipids, notably cardiolipin, and thereby enhance the release of the hemoprotein caused by treatment of mitochondria with digitonin or the proapoptotic protein Bax. Combined, our data suggest that caspase-2 possesses an unparalleled ability to engage the mitochondrial apoptotic pathway by permeabilizing the outer mitochondrial membrane and/or by breaching the association of cytochrome c with the inner mitochondrial membrane.

Caspase 2↗
PubMed2004

Processed caspase-2 can induce mitochondria-mediated apoptosis independently of its enzymatic activity.

The mechanism by which caspase-2 executes apoptosis remains obscure. Recent findings indicate that caspase-2 is activated early in response to DNA-damaging antineoplastic agents and may be important for the engagement of the mitochondrial apoptotic pathway. We demonstrate here that fully processed caspase-2 stimulates mitochondrial release of cytochrome c and Smac/DIABLO, but not apoptosis-inducing factor (AIF). This event occurs independently of several Bcl-2 family proteins, including Bax, Bak and Bcl-2, and inactivation experiments reveal that the proteolytic activity of caspase-2 is not required for the effect. Further, functional studies of mitochondria indicate that processed caspase-2 stimulates state 4 respiration and decreases the respiratory control ratio as a result of, in large part, an uncoupling effect. Combined, our data suggest that caspase-2 retains a unique ability to engage directly the mitochondrial apoptotic pathway, an effect that requires processing of the zymogen but not the associated catalytic activity.

Apoptosis↗
PubMed2004

Mitochondrial cytochrome c release may occur by volume-dependent mechanisms not involving permeability transition.

The mechanisms regulating mitochondrial outer-membrane permeabilization and the release of cytochrome c during apoptosis remain controversial. In the present study, we show in an in vitro model system that the release of cytochrome c may occur via moderate modulation of mitochondrial volume, irrespective of the mechanism leading to the mitochondrial swelling. In contrast with mitochondrial permeability transition-dependent release of cytochrome c, in the present study mitochondria remain intact and functionally active.

Animals↗
PubMed2004

Differential regulation of the mitochondrial and death receptor pathways in neural stem cells.

Despite an increasing interest in neural stem cell (NSC) research, relatively little is known about the biochemical regulation of cell death pathways in these cells. We demonstrate here, using murine-derived multipotent C17.2 NSCs, that cells undergo mitochondria-mediated cell death in response to apoptotic stimuli such as oxidative stress induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). In particular, treated cells exhibited apoptotic features, including Bax translocation, cytochrome c release, activation of caspase-9 and -3, chromatin condensation and DNA fragmentation. Although C17.2 cells possess the Fas receptor and express procaspase-8, agonistic Fas mAb treatment failed to induce apoptosis. Fas treatment activated the extracellular signal-regulated protein kinase (ERK) pathway, which may have an antiapoptotic as well as a growth stimulating role. Combined, our findings indicate that while NSCs are sensitive to cytotoxic stimuli that involve an engagement of mitochondria, Fas treatment does not induce death and may have an alternative role.

Amino Acid Chloromethyl Ketones↗
PubMed2002

Role of mitochondria in toxic cell death.

Apoptosis is a highly regulated form of cell death distinguished by the activation of a family of cysteine-aspartate proteases (caspases) that cleave various proteins resulting in morphological and biochemical changes characteristic of this form of cell death. Abundant evidence supports a role for mitochondria in regulating apoptosis. Specifically, it seems that a number of death triggers target these organelles and stimulate, by an unknown mechanism, the release of several proteins, including cytochrome c. Once released into the cytosol, cytochrome c binds to its adaptor molecule, apoptotic protease activating factor-1, which oligomerizes and then activates pro-caspase-9. Caspase-9 can signal downstream and activate pro-caspase-3 and -7. The release of cytochrome c can be influenced by different Bcl-2 family member proteins, including Bax, Bid, Bcl-2, and Bcl-X(L). Bax and Bid potentiate cytochrome c release, whereas Bcl-2 and Bcl-X(L) antagonize this event. Although toxicologists have traditionally associated cell death with necrosis, emerging evidence suggests that different types of environmental contaminants exert their toxicity, at least in part, by triggering apoptosis. The mechanism responsible for eliciting the pro-apoptotic effect of a given chemical is often unknown, although in many instances mitochondria appear to be key participants. Here, we provide an overview of our current understanding of the role of apoptosis in toxicant-induced cell death, using dioxin, organotin compounds, dithiocarbamates, as well as the chemotherapeutic agent etoposide, as specific examples.

Animals↗
PubMed2002

Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis.

DNA damage induced by the cancer chemotherapeutic drug etoposide triggers the onset of a series of intracellular events characteristic of apoptosis. Among the early changes observed is the release of cytochrome c from mitochondria, although the mechanism responsible for this effect is unclear. We demonstrate here a role for caspase-2 in etoposide-induced cytochrome c release. In particular, Jurkat T-lymphocytes treated with an irreversible caspase-2 inhibitor, benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone (z-VDVAD-fmk), or stably transfected with pro-caspase-2 antisense (Casp-2/AS) are refractory to cytochrome c release stimulated by etoposide. Experiments performed using a reconstituted cell-free system indicate that etoposide-induced cytochrome c release by way of caspase-2 occurs independently of cytosolic factors, suggesting that the nuclear pool of pro-caspase-2 is critical to this process. Apart from inhibiting cytochrome c release, undermining caspase-2 activity results in an attenuation of downstream events, such as pro-caspase-9 and -3 activation, phosphatidylserine exposure on the plasma membrane, and DNA fragmentation. Taken together, our data indicate that caspase-2 provides an important link between etoposide-induced DNA damage and the engagement of the mitochondrial apoptotic pathway.

Apoptosis↗
PubMed2002

Cytochrome c release from mitochondria proceeds by a two-step process.

Cytochrome c is often released from mitochondria during the early stages of apoptosis, although the precise mechanisms regulating this event remain unclear. In this study, with isolated liver mitochondria, we demonstrate that cytochrome c release requires a two-step process. Because cytochrome c is present as loosely and tightly bound pools attached to the inner membrane by its association with cardiolipin, this interaction must first be disrupted to generate a soluble pool of this protein. Specifically, solubilization of cytochrome c involves a breaching of the electrostatic and/or hydrophobic affiliations that this protein usually maintains with cardiolipin. Once cytochrome c is solubilized, permeabilization of the outer mitochondrial membrane by Bax is sufficient to allow the extrusion of this protein into the extramitochondrial environment. Neither disrupting the interaction of cytochrome c with cardiolipin, nor permeabilizing the outer membrane with Bax, alone, is sufficient to trigger this protein's release. This mechanism also extends to conditions of mitochondrial permeability transition insofar as cytochrome c release is significantly depressed when the electrostatic interaction between cytochrome c and cardiolipin remains intact. Our results indicate that the release of cytochrome c involves a distinct two-step process that is undermined when either step is compromised.

Animals↗