Release Of Mitochondrial Proteins Research Articles

Laser micro-irradiation of mitochondria: is there an amplified mitochondrial death signal in neural cells?

Several mitochondrial proteins, such as cytochrome c, are directly involved in the pathway for caspase activation following induction of apoptosis. Release of mitochondrial cytochrome c early in apoptosis is rapid and almost complete. Microinjection of cytochrome c into resting cells induces apoptosis, but the amount needed approaches the total cellular content. These observations suggest that mitochondrial protein release is an all-or-nothing process inside the cell and not an amplifiable apoptotic signal. To test this hypothesis, laser micro-irradiation was used to rupture membranes of individual mitochondria within living rat neural cells. Laser micro-irradiation caused swelling, fragmentation, depolarization, and cytochrome c depletion in targeted mitochondria. These effects were explained by correlative electron microscopic analysis showing local rupture of outer and inner membranes at the site of irradiation. In all cases, there were no detectable changes in the structure, membrane potential, or cytochrome c content of neighboring, non-irradiated organelles. Furthermore, irradiation of up to 15% of the mitochondria in a cell did not induce apoptosis. The results from these laser micro-irradiation experiments prove that local release of mitochondrial proteins does not constitute an amplifiable apoptotic signal in resting neural cells.

Hexokinase II: the integration of energy metabolism and control of apoptosis.

Hexokinase II is often highly expressed in poorly differentiated and rapidly growing tumors that exhibit a high rate of aerobic glycolysis. Hexokinase II binds to the mitochondrial membrane through its interaction with the outer membrane voltage-dependent anion channel (VDAC), preferentially at contact sites between the outer and inner mitochondrial membrane. This location is thought to be important for the integration of glycolysis with mitochondrial energy metabolism. VDAC is a critical component of the mitochondrial phase of apoptosis and its interaction with Bcl-2 family proteins controls the rate of release of mitochondrial intermembrane space proteins that activate the execution phase of apoptosis. The proteins involved in the contact sites also constitute the mitochondrial permeability transition, one of the mechanisms by which mitochondrial protein release can be mediated. Hexokinase II binding to VDAC suppresses the release of intermembrane space proteins and inhibits apoptosis, thereby contributing to the survival advantage of tumor cells. This interaction places hexokinase II in a position to integrate glycolytic metabolism of the tumor cell with the control of apoptosis at the mitochondrial level. Mitochondrial binding of hexokinase II may constitute an attractive target for therapeutic intervention to suppress tumor growth.

JNK-dependent Release of Mitochondrial Protein, Smac, during Apoptosis in Multiple Myeloma (MM) Cells

Smac, second mitochondria-derived activator of caspases, promotes apoptosis via activation of caspases. Previous studies have shown that c-Jun NH(2)-terminal kinase (JNK) is involved in regulating another mitochondrial protein, cytochrome c during apoptosis; however, the role of JNK in the release of mitochondrial Smac is unknown. Here we show that induction of apoptosis in multiple myeloma (MM) cells is associated with activation of JNK, translocation of JNK from cytosol to mitochondria, and release of Smac from mitochondria to cytosol. Blocking JNK either by dominant-negative mutant (DN-JNK) or cotreatment with a specific JNK inhibitor, SP600125, abrogates both stress-induced release of Smac and induction of apoptosis. These findings demonstrate that activation of JNK is an obligatory event for the release of Smac during stress-induced apoptosis in MM cells.

Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells.

Cytokines such as Fas-ligand (Fas-L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) can induce human colon cancer cell apoptosis through engagement of their death domain receptors. All the cancer cells are not sensitive to these cytokines. We have shown recently that low doses of cytotoxic drugs could restore TRAIL-induced cell death in resistant colon cancer cell lines. The present work further explores the death pathway triggered by the cytotoxic drug/TRAIL combination in HT-29 colon cancer cells (www.alexis-corp.com). Clinically relevant concentrations of cisplatin, doxorubicin and 5-fluorouracil synergize with TRAIL to trigger HT-29 cell death. Activation of this pathway leads to apoptosis that involves both caspases and the mitochondria. An increased recruitment of Fas-associated death domain (FADD) and procaspase-8 to the TRAIL-induced death-inducing signaling complex (DISC) was shown in cells exposed to anticancer drugs. Following caspase-8 activation at the DISC level, the mitochondria-dependent death pathway is activated, as demonstrated by the cleavage of Bid, the dissipation of DeltaPsi(m), the release of mitochondrial proteins in the cytosol and the inhibitory effect of Bcl-2 expression. Importantly, besides mitochondrial potentiation, we show here that cytotoxic drugs sensitize HT-29 colon cancer cells to TRAIL-induced cell death by enhancing FADD and procaspase-8 recruitment to the DISC, a novel mechanism whose efficacy could depend partly on Bcl-2 expression level.

Bid, Bax, and Lipids Cooperate to Form Supramolecular Openings in the Outer Mitochondrial Membrane

Bcl-2 family proteins regulate the release of proteins like cytochrome c from mitochondria during apoptosis. We used cell-free systems and ultimately a vesicular reconstitution from defined molecules to show that outer membrane permeabilization by Bcl-2 family proteins requires neither the mitochondrial matrix, the inner membrane, nor other proteins. Bid, or its BH3-domain peptide, activated monomeric Bax to produce membrane openings that allowed the passage of very large (2 megadalton) dextran molecules, explaining the translocation of large mitochondrial proteins during apoptosis. This process required cardiolipin and was inhibited by antiapoptotic Bcl-x L. We conclude that mitochondrial protein release in apoptosis can be mediated by supramolecular openings in the outer mitochondrial membrane, promoted by BH3/Bax/lipid interaction and directly inhibited by Bcl-x L.

Novel strategies for overcoming multidrug resistance in cancer.

The problems of why metastatic cancers develop pleiotropic resistance to all available therapies, and how this might be countered, are the most pressing in cancer chemotherapy. It is likely that such resistance involves a combination of mechanisms including changes in drug transport/drug targets, reduction in the degree of drug-induced apoptosis/cell loss, and increased rate of tumour repopulation following therapy. Current research must consider not only which mechanisms contribute, eventually relating this to individual patients with cancer, but also what strategies might be utilised to counter each of the important resistance mechanisms. A considerable amount of work has been devoted to the development of inhibitors of membrane-associated transport proteins such as P-glycoprotein, which mediate drug efflux. This work is now being complemented by approaches that target cell death pathways such as those mediated by release of mitochondrial proteins and by activation of surface receptors such as Fas. Rapid progress has been made in developing small-molecular-weight drugs that influence the rate of apoptosis, for instance by binding to the bcl-2 family of proteins regulating mitochondrial permeability. Antisense approaches aimed at reducing bcl-2 expression, and thus increasing the rate of cell death, are also showing promise. Modification of repopulation kinetics provides a further approach but has not received as much attention as other aspects of tumour resistance. New therapeutic approaches will have to be complemented by improved diagnostic tests to evaluate the contributions of different resistance mechanisms in individual patients with cancer.

Mitochondria in muscle cell death.

Mitochondria, the main source of energy for eukaryotic cells through oxidative phosphorylation, also play a key role in the pathways to cell death. The mode of cell death may be influenced by the availability of ATP, and its very occurrence may critically depend on release of mitochondrial proteins like cytochrome c, apoptosis-inducing factor and possibly caspases 3 and 9. Ca2+-dependent onset of the permeability transition, caused by opening of a cyclosporin A-sensitive pore modulated by cyclophilin D, may play a major role in cell death through ATP depletion, disruption of Ca2+ homeostasis, and release of specific mitochondrial proteins. Dysregulation of Ca2+ homeostasis, proteolysis and a decreased ability to cope with oxidative stress are involved in the pathogenesis of Duchenne's muscular dystrophy downstream of the genetic lesion, and mitochondria appear as likely targets that may amplify the initial insult resulting in the irreversible events leading to cell demise. My colleagues and I are studying the permeability transition in skeletal muscle mitochondria, and we are validating bupivacaine in a short-term model of muscle cell toxicity involving mitochondrial depolarization and pore opening as early events. Specific goals for the future are to further define the role of mitochondria in muscle cell death, with particular emphasis on the role of the permeability transition pore and cyclophilin D, and to develop and test drugs able to affect its course in model systems in vitro and in the mdx mouse, an animal model of Duchenne's muscular dystrophy.

Fas ligand, death gene.

The concept of death genes goes back to the early days of programmed cell death, when a researcher's model system was required to be dependent on transcription of the dying cell in order to qualify as apoptosis. In 1987 Andrew Wyllie,1 one of the pioneers of cell death research, outlined four 'cardinal elements' of apoptosis: one of which was a requirement for macromolecular synthesis. In the following years the complexity of the apoptotic process has become evident and while it is now clear that apoptosis does not have to rely on gene expression, the idea of death genes remains. Induction of an apoptotic cascade via activation of caspases, selective release of mitochondrial proteins and further activation of caspases, can be stimulated by engagement of the Fas surface molecule via membrane bound or soluble forms of Fas ligand (FasL). The FasL gene, which is often transcriptionally inactive, becomes activated in many forms of transcription/translation dependent apoptosis. Here we will discuss FasL as a candidate death gene.

Role and mechanisms of secondary mitochondrial failure.

Ischemia is accompanied by mitochondrial dysfunction, as assessed by measurements of mitochondrial respiratory activities in vitro. Following brief periods of ischemia, mitochondrial function is usually normalized during reperfusion. However, particularly after ischemia of longer duration, reperfusion may be accompanied by secondary mitochondrial failure. After short periods of ischemia this is observed in selectively vulnerable areas and, after intermediate to long periods of ischemia, in other areas as well. However, it has remained unsettled if the mitochondrial dysfunction is the result or the cause of cell death. Although it has been commonly assumed that such failure is secondary to cell injury by other mechanisms, recent results suggest that mitochondrial dysfunction may be the cause of cell death. Indirect evidence for this postulate is provided by experiments showing that cyclosporin A (CsA), when allowed to cross the blood-brain barrier, is a potent neuroprotectant. CsA is a virtually specific blocker of the mitochondrial permeability transition (MPT) pore, a voltage-gated channel allowing molecules and ions with a mass < 1500 Daltons to pass the inner mitochondrial membrane. Experiments on isolated cells in vitro demonstrate that cell calcium accumulation or oxidative stress triggers the assembly of an MPT pore, which leads to collapse of the mitochondrial membrane potential, to ATP hydrolysis, to enhanced production of reactive oxygen species (ROS), and to cell death. The beneficial effect of CsA could thus be related to its ability to block the MPT pore. Longer periods of ischemia, such as occurs after transient middle cerebral artery (MCA) occlusion, lead to pan-necrotic lesions (infarction). In the rat, recirculation following 2 h of MCA occlusion leads to partial normalization of the bioenergetic state but this is followed within 4-6 h by secondary bioenergetic failure. The latter seems unrelated to blockade of the microcirculation, but correlates to secondary mitochondrial failure. The brain damage incurred is ameliorated by the spin trap alpha-phenyl-N-butyl nitrone (PBN) and by the immunosuppressant FK506 even when given 1-3 h after the start of recirculation. The two drugs also prevent the secondary mitochondrial failure during early recirculation, suggesting that such failure is pathogenetically important. Probably, though, the mitochondrial dysfunction involves not only the assembly of an MPT pore but also other mechanisms. Since recirculation is associated with release of mitochondrial proteins it is not unlikely that such proteins, e.g. cytochrome c, trigger cascades of events leading to cell death.6.

Recent Progress on the Regulation of Apoptosis by Bcl-2 Family Members

Publisher Summary Programmed cell death (PCD) plays a pivotal role in the development, in the maintenance of homeostasis within multicellular organisms, and mutations in genes known to control PCD have been associated with pathologies, such as cancer, autoimmunity, and neurodegenerative disorders. A family of critical cell death regulators known as the Bcl-2 family is described along with some of the most recent advances in the understanding of their properties, structure, and function. Although apoptosis is a complex process, the available data suggest that Bcl-2 family members regulate a focal point, where a motley of upstream apoptotic signals first converge, and may regulate caspase activation by preventing alterations in mitochondrial homeostasis that would otherwise lead to the release of mitochondrial proteins that are capable of activating downstream caspases. The formation of ion channels by Bcl-2 family members may be one mechanism by which these proteins control mitochondrial processes. In addition to the multitude of proteins that are associated with Bcl-2 family members, many other interesting proteins, such as Raf-1 can also associate and, in some cases, influence the cell survival. Future studies are needed to test the importance of the biochemical activities identified for members of the Bcl-2 family, to determine the relative importance of the interactions between Bcl-2 family members and the proteins they interact with, and how these protein interactions regulate function.

Release of mitochondrial aspartate aminotransferase (mAST) following transient focal cerebral ischemia suggests the opening of a mitochondrial permeability transition pore

The present experiments were undertaken to study if ischemia and reperfusion in the brain are accompanied by a mitochondrial permeability transition (MPT), leading to the release of mitochondrial proteins into the cytosol. The protein studied was the mitochondrial isoform of aspartate aminotransferase (mAST). In vitro experiments showed that isolated brain mitochondria exposed to calcium (50 μM) and phosphate (0.5 mM) displayed rapidly developing swelling, as evidenced by a change in light scattering at 540 nm. The swelling which could be reversal by chelation of external calcium, was associated with release of mAST. Focal ischemia of 2 h duration was induced by occlusion of one middle cerebral artery (MCA) by an intraluminal filament technique. The ischemia gave rise to a marked decrease in respiratory control ratio (RCR) of mitochondria in the homogenate. The RCR recovered partly after 1 h of recirculation, but decrease again after 4 h. The mAST in the cytosolic fraction did not change at the end of the ischemia, but increased significantly after 1 h of recirculation in the focus of the lesion, but not in the penumbra. Little further change occurred thereafter (4 h). It is concluded that in densely ischemic areas, recirculation yields rapidly developing dysfunction of the inner mitochondrial membrane, with release of mitochondrial proteins. © 1997 John Wiley & Sons, Ltd.

Adenosine 5'-triphosphate stimulates the release of polypeptides from mitochondria

There was release of polypeptides to the medium when mitochondria containing labeled proteins were incubated with a rat liver post-mitochondrial supernatant. The release of polypeptides increased with the amount of rat liver extract added. Addition of cycloheximide did not inhibit the effect. Heating the postmitochondrial supernatant did not inhibit the release of mitochondrial proteins, indicating that it was due to a heat-stable factor. The factor responsible has been isolated and identified as ATP. The presence of EDTA inhibits the release of polypeptides caused by ATP and Mg 2+ stimulates it. The possible role of ATP in the turnover of mitochondrial proteins is briefly discussed. Adenosine 5'-triphosphate Mitochondria Protein turnover

An ammonium sulphate fraction from rabbit reticulocytes increases the release of proteins from rat liver mitochondria

Incubation of [ 35S]methionine labeled mitochondria from rat liver with rabbit reticulocyte lysate under the same conditions as those used in the import of mitochondrial protein precursors results in the release of mitochondrial proteins to the medium. Fractionation of the lysates with ammonium sulphate yields a fraction, essentially free of haemoglobin, which exhibits higher activity for the release of mitochondrial proteins than the starting lysate. The fraction has a molecular mass of > 10 kDa and is heat-sensitive. The release is insensitive to inhibitors of reticulocyte lipoxygenase.

Effect of exogenous addition of hemin on the biogenesis of mitochondrial membranes during glucose repression in Saccharomyces cerevisiae

Exogenous addition of hemin alleviated glucose repression by promoting mitochondrial membrane functions. It prevented the release of mitochondrial proteins into the cytosol by decreasing the activities of phospholipase C and phospholipase D. It restored oligomycin sensitivity of mitochondrial ATPase associated with repressed mitochondria. It enhanced cardiolipin content twofold, and decreased the sterol: phospholipid ratio. Studies on the amino acid incorporation into isolated mitochondria showed that hemin can also promote biogenesis of the organelle by stimulating amino acid incorporation into mitochondrial proteins, and this stimulation appeared to be mediated through some cytosolic factor(s).

Exit of proteins and fragments thereof from mitochondria is accelerated by the import of cytosolic synthesized proteins

Most mitochondrial proteins are synthesized on cytosolic ribosomes and imported into mitochondria. Incubation of 35S-methionine labeled mitochondria from rat hepatocytes with proteins synthesized in a cell-free system, using messenger RNA from rat liver, dramatically increased the release of mitochondrial proteins and fragments thereof into the medium. Since the synthesized proteins include cytosolic precursors of mitochondrial proteins, our results strongly suggest that import of proteins from the cytosol into mitochondria influences the half-life of proteins in these organelles. The use of this simple approach — i.e. combining the study of protein import and exit with mitochondria — to further clarify intracellular protein turnover and its regulation is suggested.