Bcl-2 regulates amplification of caspase activation by cytochrome c.
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Caspases, a family of specific proteases, have central roles in apoptosis [1]. Caspase activation in response to diverse apoptotic stimuli involves the relocalisation of cytochrome c from mitochondria to the cytoplasm where it stimulates the proteolytic processing of caspase precursors. Cytochrome c release is controlled by members of the Bcl-2 family of apoptosis regulators [2] [3]. The anti-apoptotic members Bcl-2 and Bcl-xL may also control caspase activation independently of cytochrome c relocalisation or may inhibit a positive feedback mechanism [4] [5] [6] [7]. Here, we investigate the role of Bcl-2 family proteins in the regulation of caspase activation using a model cell-free system. We found that Bcl-2 and Bcl-xL set a threshold in the amount of cytochrome c required to activate caspases, even in soluble extracts lacking mitochondria. Addition of dATP (which stimulates the procaspase-processing factor Apaf-1 [8] [9]) overcame inhibition of caspase activation by Bcl-2, but did not prevent the control of cytochrome c release from mitochondria by Bcl-2. Cytochrome c release was accelerated by active caspase-3 and this positive feedback was negatively regulated by Bcl-2. These results provide evidence for a mechanism to amplify caspase activation that is suppressed at several distinct steps by Bcl-2, even after cytochrome c is released from mitochondria. (+info)
Hsp60 is targeted to a cryptic mitochondrion-derived organelle ("crypton") in the microaerophilic protozoan parasite Entamoeba histolytica.
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Entamoeba histolytica is a microaerophilic protozoan parasite in which neither mitochondria nor mitochondrion-derived organelles have been previously observed. Recently, a segment of an E. histolytica gene was identified that encoded a protein similar to the mitochondrial 60-kDa heat shock protein (Hsp60 or chaperonin 60), which refolds nuclear-encoded proteins after passage through organellar membranes. The possible function and localization of the amebic Hsp60 were explored here. Like Hsp60 of mitochondria, amebic Hsp60 RNA and protein were both strongly induced by incubating parasites at 42 degreesC. 5' and 3' rapid amplifications of cDNA ends were used to obtain the entire E. histolytica hsp60 coding region, which predicted a 536-amino-acid Hsp60. The E. histolytica hsp60 gene protected from heat shock Escherichia coli groEL mutants, demonstrating the chaperonin function of the amebic Hsp60. The E. histolytica Hsp60, which lacked characteristic carboxy-terminal Gly-Met repeats, had a 21-amino-acid amino-terminal, organelle-targeting presequence that was cleaved in vivo. This presequence was necessary to target Hsp60 to one (and occasionally two or three) short, cylindrical organelle(s). In contrast, amebic alcohol dehydrogenase 1 and ferredoxin, which are bacteria-like enzymes, were diffusely distributed throughout the cytosol. We suggest that the Hsp60-associated, mitochondrion-derived organelle identified here be named "crypton," as its structure was previously hidden and its function is still cryptic. (+info)
An alternative transcript of the rat renin gene can result in a truncated prorenin that is transported into adrenal mitochondria.
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Characterization of the local renin-angiotensin system in the rat adrenal zona glomerulosa indicated a dual targeting of renin both to the secretory pathway and mitochondria. To investigate the transport of renin into mitochondria, we constructed a series of amino-terminal deletion variants of preprorenin. One of these variants, lacking the complete signal sequence for the endoplasmic reticulum and 10 amino acids of the profragment, was transported efficiently into isolated mitochondria. The transport was further shown to be dependent on mitochondrial membrane potential and ATP synthesis. Analysis of adrenal RNA revealed the existence of 2 renin transcripts. While one of the transcripts corresponds to the known full-length transcript, the other one lacks exon 1; instead, exon 2 is preceded by a domain of 80 nucleotides originating from intron 1. This domain, as well as the following region of intron 1 being excised, shows all essential sequence elements defining an additional, so-far-unknown exon. The second mRNA possibly derives from an additional transcription start in intron 1 and an alternative splicing process. Translation of this mRNA could result in a truncated prorenin representing a cytosolic form of renin, which is required for transport into mitochondria. This truncated prorenin corresponds exactly to the deletion variant being imported into mitochondria in vitro. (+info)
UCP4, a novel brain-specific mitochondrial protein that reduces membrane potential in mammalian cells.
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Uncoupling proteins (UCPs) are a family of mitochondrial transporter proteins that have been implicated in thermoregulatory heat production and maintenance of the basal metabolic rate. We have identified and partially characterized a novel member of the human uncoupling protein family, termed uncoupling protein-4 (UCP4). Protein sequence analyses showed that UCP4 is most related to UCP3 and possesses features characteristic of mitochondrial transporter proteins. Unlike other known UCPs, UCP4 transcripts are exclusively expressed in both fetal and adult brain tissues. UCP4 maps to human chromosome 6p11.2-q12. Consistent with its potential role as an uncoupling protein, UCP4 is localized to the mitochondria and its ectopic expression in mammalian cells reduces mitochondrial membrane potential. These findings suggest that UCP4 may be involved in thermoregulatory heat production and metabolism in the brain. (+info)
R73A and H144Q mutants of the yeast mitochondrial cyclophilin Cpr3 exhibit a low prolyl isomerase activity in both peptide and protein-folding assays.
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Previously we reported that the R73A and H144Q variants of the yeast cyclophilin Cpr3 were virtually inactive in a protease-coupled peptide assay, but retained activity as catalysts of a proline-limited protein folding reaction [Scholz, C. et al. (1997) FEBS Lett. 414, 69-73]. A reinvestigation revealed that in fact these two mutations strongly decrease the prolyl isomerase activity of Cpr3 in both the peptide and the protein-folding assay. The high folding activities found previously originated from a contamination of the recombinant Cpr3 proteins with the Escherichia coli protein SlyD, a prolyl isomerase that co-purifies with His-tagged proteins. SlyD is inactive in the peptide assay, but highly active in the protein-folding assay. (+info)
Mitochondrial depolarization accompanies cytochrome c release during apoptosis in PC6 cells.
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Cytochrome c is released from mitochondria into the cytosol in cells undergoing apoptosis. The temporal relationship between cytochrome c release and loss of mitochondrial membrane potential was monitored by laser-scanning confocal microscopy in single living pheochromocytoma-6 cells undergoing apoptosis induced by staurosporine. Mitochondrial membrane potential monitored by tetramethylrhodamine methyl ester decreased abruptly in individual cells from 2 to 7 h after treatment with staurosporine. Depolarization was accompanied by cytochrome c release documented by release of transfected green fluorescent protein-tagged cytochrome c in these cells. The results show that mitochondrial depolarization accompanies cytochrome c release in pheochromocytoma-6 cells undergoing apoptosis. (+info)
Identification of 17-methyl-18-norandrosta-5,13(17-dien-3beta-ol, the C19 fragment formed by adrenal side chain cleavage of a 20-aryl analog of (20S)-20-hydroxycholesterol.
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Incubation of (20R)-20-phenyl-5-pregnene-3beta,20-diol, an aromatic analog of (23S)-20-hydroxycholesterol, with an adrenal mitochondrial preparation leads to the formation of four compounds: pregnenolone, phenol, a C8 ketone, acetophenone, and a nonpolar C19 compound. This latter compound has now been identified by reverse isotope dilution analysis and by gas chromatography/mass spectrometry as 17-methyl-18-norandrosta-5,13(17)-dien-3beta-ol. From these results it is evident that enzymatic fission of the C-17,20 bond of this synthetic derivative occurs. On the other hand, when (20S)-20-hydroxy[21-14C]cholesterol was used as substrate, the analogous cleavage did not take place. Thus, substitution of an aromatic group on C-20 facilitates side chain cleavage between that carbon atom and the nucleus whereas neither of the naturally occuring precursors, cholesterol or its 20-hydroxylated counterpart, are metabolized to a C8 fragment. (+info)
Role of hypoxia-induced Bax translocation and cytochrome c release in reoxygenation injury.
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We investigated mechanisms of cell death during hypoxia/reoxygenation of cultured kidney cells. During glucose-free hypoxia, cell ATP levels declined steeply resulting in the translocation of Bax from cytosol to mitochondria. Concurrently, there was cytochrome c release and caspase activation. Cells that leaked cytochrome c underwent apoptosis after reoxygenation. ATP depletion induced by a mitochondrial uncoupler resulted in similar alterations even in the presence of oxygen. Moreover, inclusion of glucose during hypoxia prevented protein translocations and reoxygenation injury by maintaining intracellular ATP. Thus, ATP depletion, rather than hypoxia per se, was the cause of protein translocations. Overexpression of Bcl-2 prevented cytochrome c release and reoxygenation injury without ameliorating ATP depletion or Bax translocation. On the other hand, caspase inhibitors did not prevent protein translocations, but inhibited apoptosis during reoxygenation. Nevertheless, they could not confer long-term viability, since mitochondria had been damaged. Omission of glucose during reoxygenation resulted in continued failure of ATP production, and cell death with necrotic morphology. In contrast, cells expressing Bcl-2 had functional mitochondria and remained viable during reoxygenation even without glucose. Therefore, Bax translocation during hypoxia is a molecular trigger for cell death during reoxygenation. If ATP is available during reoxygenation, apoptosis develops; otherwise, death occurs by necrosis. By preserving mitochondrial integrity, BCL-2 prevents both forms of cell death and ensures cell viability. (+info)