Oma1, a novel membrane-bound metallopeptidase in mitochondria with activities overlapping with the m-AAA protease

The integrity of the inner membrane of mitochondria is maintained by a membrane-embedded quality control system that ensures the removal of misfolded membrane proteins. Two ATP-dependent AAA proteases with catalytic sites at opposite membrane surfaces are key components of this proteolytic system. Here we describe the identification of a novel conserved metallopeptidase that exerts activities overlapping with the m-AAA protease and was therefore termed Oma1. Both peptidases are integral parts of the inner membrane and mediate the proteolytic breakdown of a misfolded derivative of the polytopic inner membrane protein Oxa1. The m-AAA protease cleaves off the matrix-exposed C-terminal domain of Oxa1 and processively degrades its transmembrane domain. In the absence of the m-AAA protease, proteolysis of Oxa1 is mediated in an ATP-independent manner by Oma1 and a yet unknown peptidase resulting in the accumulation of N- and C-terminal proteolytic fragments. Oma1 exposes its proteolytic center to the matrix side; however, mapping of Oma1 cleavage sites reveals clipping of Oxa1 in loop regions at both membrane surfaces. These results identify Oma1 as a novel component of the quality control system in the inner membrane of mitochondria. Proteins homologous to Oma1 are present in higher eukaryotic cells, eubacteria and archaebacteria, suggesting that Oma1 is the founding member of a conserved family of membrane-embedded metallopeptidases.     

12(S)-hydroperoxyeicosatetraenoic acid (12-HETE) increases mitochondrial nitric oxide by increasing intramitochondrial calcium

12(S)-Hydroxyeicosatetraenoic acid (12-HETE) is one of the metabolites of arachidonic acid involved in pathological conditions associated with mitochondria and oxidative stress. The present study tested effects of 12-HETE on mitochondrial functions. In isolated rat heart mitochondria, 12-HETE increases intramitochondrial ionized calcium concentration that stimulates mitochondrial nitric oxide (NO) synthase (mtNOS) activity. mtNOS-derived NO causes mitochondrial dysfunctions by decreasing mitochondrial respiration and transmembrane potential. mtNOS-derived NO also produces peroxynitrite that induces release of cytochrome c and stimulates aggregation of mitochondria. Similarly, in HL-1 cardiac myocytes, 12-HETE increases intramitochondrial calcium and mitochondrial NO, and induces apoptosis. The present study suggests a novel mechanism for 12-HETE toxicity.     

Selective Oma1 protease-mediated proteolysis of Cox1 subunit of cytochrome oxidase in assembly mutants

Stalled biogenesis of the mitochondrial cytochrome c oxidase (CcO) complex results in degradation of subunits containing redox cofactors. The conserved Oma1 metalloproteinase mediates facile Cox1 degradation in cells lacking the Coa2 assembly factor, but not in a series of other mutants stalled in CcO maturation. Oma1 is activated in coa2Δ cells, but the selective Cox1 degradation does not arise merely from its activation. Oma1 is also active in cells with dysfunctional mitochondria and cox11Δ cells impaired in CcO maturation, but this activation does not result in Oma1-mediated Cox1 degradation. The facile and selective degradation of Cox1 in coa2Δ cells, relative to other CcO assembly mutants, is likely due to impaired hemylation and subsequent misfolding of the subunit. Specific Cox1 proteolysis in coa2Δ cells arises from a combination of Oma1 activation and a susceptible conformation of Cox1.     

Direct antioxidant and protective effect of estradiol on isolated mitochondria

Estrogens have antioxidant properties which are due to their ability to bind to estrogen receptors and to up-regulate the expression of antioxidant enzymes via intracellular signalling pathways. Mitochondria are key organelles in the development of age-associated cellular damage. Recently, estrogen receptors were identified in mitochondria. The aim of this paper was to test whether estradiol directly affects mitochondria by preventing oxidative stress and protecting frail mitochondria. Incubation with estradiol at normal intracellular concentrations prevents the formation of reactive oxygen species by mitochondria in a saturable manner. Moreover, estradiol protects mitochondrial integrity as indicated by an increase in mitochondrial membrane potential. It also prevents the apoptogenic leakage of cytochrome c from mitochondria and as a result the mitochondrial content of this cytochrome c is maintained high. Thus, estradiol prevents the onset of the mitochondrial pathway of apoptosis by a direct effect on the organelle. Genistein, a phytoestrogen present at high concentration in soy, mimics the protective effect of estradiol by both decreasing the rate of formation of reactive oxygen species and preventing the release of cytochrome c from mitochondria.     

Cox17 Protein Is an Auxiliary Factor Involved in the Control of the Mitochondrial Contact Site and Cristae Organizing System

The mitochondrial contact site and cristae organizing system (MICOS) is a recently discovered protein complex that is crucial for establishing and maintaining the proper inner membrane architecture and contacts with the outer membrane of mitochondria. The ways in which the MICOS complex is assembled and its integrity is regulated remain elusive. Here, we report a direct link between Cox17, a protein involved in the assembly of cytochrome c oxidase, and the MICOS complex. Cox17 interacts with Mic60, thereby modulating MICOS complex integrity. This interaction does not involve Sco1, a partner of Cox17 in transferring copper ions to cytochrome c oxidase. However, the Cox17-MICOS interaction is regulated by copper ions. We propose that Cox17 is a newly identified factor involved in maintaining the architecture of the MICOS complex.     

Mitochondrial-targeted active Akt protects SH-SY5Y neuroblastoma cells from staurosporine-induced apoptotic cell death

Akt is a serine/threonine protein kinase that plays a vital role in promoting cellular survival. Predominantly cytosolic, upon stimulation with growth-factors or stress, active Akt translocates into mitochondria, but the functions of Akt in mitochondria are not yet fully understood. Mitochondria play a central role in apoptotic pathways and given Akt's functions in the cytoplasm, Akt in mitochondria may help preserve mitochondrial integrity during cellular stress. To test if the translocation of Akt into mitochondria is neuroprotective, adenoviral vectors expressing a constitutively active Akt, Ad-HA-Akt (DD), and a constitutively active Akt with a mitochondrial targeting signal, Ad-Mito-HA-Akt (DD), were generated. Human SH-SY5Y neuroblastoma cells expressing the adenoviral constructs were treated with staurosporine to initiate intrinsic apoptotic cell death and several aspects of the mitochondrial apoptotic pathway were evaluated. Expression of active Akt targeted to mitochondria was found to be sufficient to significantly reduce staurosporine-induced activation of caspase-3 and caspase-9, the release of cytochrome c from mitochondria, and Bax oligomerization at mitochondria. These findings demonstrate that intramitochondrial active Akt results in efficient protection against apoptotic signaling.     

Melatonin inhibits cardiolipin peroxidation in mitochondria and prevents the mitochondrial permeability transition and cytochrome c release

Cardiolipin oxidation is emerging as an important factor in mitochondrial dysfunction as well as in the initial phase of the apoptotic process. We have previously shown that exogenously added peroxidized cardiolipin sensitizes mitochondria to Ca²⁺-induced mitochondrial permeability transition (MPT) pore opening and promotes the release of cytochrome c. In this work, the effects of intramitochondrial cardiolipin peroxidation on Ca²⁺-induced MPT and on the cytochrome c release from mitochondria were studied. The effects of melatonin, a compound known to protect the mitochondria from oxidative damage, on both of these processes were also tested. tert-Butylhydroperoxide (t-BuOOH), a lipid-soluble peroxide that promotes lipid peroxidation, was used to induce intramitochondrial cardiolipin peroxidation. Exposure of heart mitochondria to t-BuOOH resulted in the oxidation of cardiolipin, associated with an increased sensitivity of mitochondria to Ca²⁺-induced MPT and with the release of cytochrome c from the mitochondria. All these processes were inhibited by micromolar concentrations of melatonin. It is proposed that melatonin inhibits cardiolipin peroxidation in mitochondria, and this effect seems to be responsible for the protection afforded by this agent against the MPT induction and cytochrome c release. Thus, manipulating the oxidation sensitivity of cardiolipin with melatonin may help to control MPT and cytochrome c release, events associated with cell death, and thus, be used for treatment of those disorders characterized by mitochondrial cardiolipin oxidation and Ca²⁺ overload.     

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die¹. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm². The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death^3-4.The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria⁵. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells^6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis^10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.     

Protein degradation in mitochondria

The biogenesis of mitochondria and the maintenance of mitochondrial functions depends on an autonomous proteolytic system in the organelle which is highly conserved throughout evolution. Components of this system include processing peptidases and ATP-dependent proteases, as well as molecular chaperone proteins and protein complexes with apparently regulatory functions. While processing peptidases mediate maturation of nuclear-encoded mitochondrial preproteins, quality control within various subcompartments of mitochondria is ensured by ATP-dependent proteases which selectively remove non-assembled or misfolded polypeptides. Moreover; these proteases appear to control the activity- or steady-state levels of specific regulatory proteins and thereby ensure mitochondrial genome integrity, gene expression and protein assembly.     

Isolating the segment of the mitochondrial electron transport chain responsible for mitochondrial damage during cardiac ischemia

Ischemia damages the mitochondrial electron transport chain (ETC), mediated in part by damage generated by the mitochondria themselves. Mitochondrial damage resulting from ischemia, in turn, leads to cardiac injury during reperfusion. The goal of the present study was to localize the segment of the ETC that produces the ischemic mitochondrial damage. We tested if blockade of the proximal ETC at complex I differed from blockade distal in the chain at cytochrome oxidase. Isolated rabbit hearts were perfused for 15 min followed by 30 min stop-flow ischemia at 37 °C. Amobarbital (2.5 mM) or azide (5 mM) was used to block proximal (complex I) or distal (cytochrome oxidase) sites in the ETC. Time control hearts were buffer-perfused for 45 min. Subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) were isolated. Ischemia decreased cytochrome c content in SSM but not in IFM compared to time control. Blockade of electron transport at complex I preserved the cytochrome c content in SSM. In contrast, blockade of electron transport at cytochrome oxidase with azide did not retain cytochrome c in SSM during ischemia. Since blockade of electron transport at complex III also prevented cytochrome c loss during ischemia, the specific site that elicits mitochondrial damage during ischemia is likely located in the segment between complex III and cytochrome oxidase.     

Studies on the orientations of the mitochondrial redox carriers II. Orientation of the mitochondrial chromophores with respect to the plane of the membrane in hydrated,oriented mitochondrial multilayers

Orientations of the active site chromophores of the mitochondrial redox carriers have been investigated in hydrated, oriented multilayers of mitochondrial membranes using optical and EPR spectroscopy. The hemes of cytochrome c oxidase, cytochrome c₁, and cytochromes b were found to be oriented in a similar manner, with the normal to their heme planes lying approximately in the plane of the mitochondrial membrane. The heme of cytochrome c was either less oriented in general or was oriented at an angle closer to the plane of the mitochondrial membrane than were the hemes of the “tightly bound” mitochondrial cytochromes. EPR spectra of the azide, sulfide and formate complexes of cytochrome c oxidase in mitochondria in situ obtained as a function of the orientation of the applied magnetic field relative to the planes of the membrane multilayers showed that both hemes of the oxidase were oriented in such a way that the angle between the heme normal and the membrane normal was approx. 90°.     

Cytochrome c interactions with membranes. A photoaffinity-labeled cytochrome c.

The aryl azide, 2,4-dinitro-5-fluorophenylazide, was reacted with horse heart cytochrome c to give a photoaffinity-labeled derivative of this heme protein. The modified cytochrome c, with one to two dinitroazidophenyl groups per mole of the enzyme, has a half-reduction potential the same (± 10 mV) as native cytochrome c. The dissociation constant for the modified cytochrome c from cytochrome c-depleted mitochondrial membranes and the apparent K_m for the reaction with cytochrome c oxidase were each five to six times greater than the values for native cytochrome c. Irradiation of cytochrome c-depleted mitochondrial membranes supplemented with an excess of photoaffinity-labeled cytochrome c resulted in covalent binding of the derivative to the mitochondrial membranes. Fractionation of the irradiated mitochondria in the presence of detergents and salts followed by chromatography on agarose, Bio-Gel A, showed that labeled cytochrome c was bound covalently to cytochrome c oxidase in a 1:1 molar complex. The covalently linked cytochrome c-cytochrome c oxidase complex was active in mediating the electron transfer between N,N,N′,N′-tetramethyl-p-phenylenediamine/ascorbate and the oxidase.     

The m-AAA Protease Processes Cytochrome c Peroxidase Preferentially at the Inner Boundary Membrane of Mitochondria

The m-AAA protease is a conserved hetero-oligomeric complex in the inner membrane of mitochondria. Recent evidence suggests a compartmentalization of the contiguous mitochondrial inner membrane into an inner boundary membrane (IBM) and a cristae membrane (CM). However, little is known about the functional differences of these subdomains. We have analyzed the localizations of the m-AAA protease and its substrate cytochrome c peroxidase (Ccp1) within yeast mitochondria using live cell fluorescence microscopy and quantitative immunoelectron microscopy. We find that the m-AAA protease is preferentially localized in the IBM. Likewise, the membrane-anchored precursor form of Ccp1 accumulates in the IBM of mitochondria lacking a functional m-AAA protease. Only upon proteolytic cleavage the mature form mCcp1 moves into the cristae space. These findings suggest that protein quality control and proteolytic activation exerted by the m-AAA protease take place preferentially in the IBM pointing to significant functional differences between the IBM and the CM.     

Human IAP-Like Protein Regulates Programmed Cell Death Downstream of Bcl-xL and Cytochrome c

The gene encoding human IAP-like protein (hILP) is one of several mammalian genes with sequence homology to the baculovirus inhibitor-of-apoptosis protein (iap) genes. Here we show that hILP can block apoptosis induced by a variety of extracellular stimuli, including UV light, chemotoxic drugs, and activation of the tumor necrosis factor and Fas receptors. hILP also protected against cell death induced by members of the caspase family, cysteine proteases which are thought to be the principal effectors of apoptosis. hILP and Bcl-x_L were compared for their ability to affect several steps in the apoptotic pathway. Redistribution of cytochrome c from mitochondria, an early event in apoptosis, was not blocked by overexpression of hILP but was inhibited by Bcl-x_L. In contrast, hILP, but not Bcl-x_L, inhibited apoptosis induced by microinjection of cytochrome c. These data suggest that while Bcl-x_L may control mitochondrial integrity, hILP can function downstream of mitochondrial events to inhibit apoptosis.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.     

The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission

Mitochondrial fusion and structure depend on the dynamin-like GTPase OPA1, whose activity is regulated by proteolytic processing. Constitutive OPA1 cleavage by YME1L and OMA1 at two distinct sites leads to the accumulation of both long and short forms of OPA1 and maintains mitochondrial fusion. Stress-induced OPA1 processing by OMA1 converts OPA1 completely into short isoforms, inhibits fusion, and triggers mitochondrial fragmentation. Here, we have analyzed the function of different OPA1 forms in cells lacking YME1L, OMA1, or both. Unexpectedly, deletion of Oma1 restored mitochondrial tubulation, cristae morphogenesis, and apoptotic resistance in cells lacking YME1L. Long OPA1 forms were sufficient to mediate mitochondrial fusion in these cells. Expression of short OPA1 forms promoted mitochondrial fragmentation, which indicates that they are associated with fission. Consistently, GTPase-inactive, short OPA1 forms partially colocalize with ER–mitochondria contact sites and the mitochondrial fission machinery. Thus, OPA1 processing is dispensable for fusion but coordinates the dynamic behavior of mitochondria and is crucial for mitochondrial integrity and quality control.     

p53 Is Required for Cisplatin-induced Processing of the Mitochondrial Fusion Protein L-Opa1 That Is Mediated by the Mitochondrial Metallopeptidase Oma1 in Gynecologic Cancers

Mitochondria are highly dynamic organelles, and mitochondrial fission is a crucial step of apoptosis. Although Oma1 is believed to be responsible for long form Opa1 (L-Opa1) processing during mitochondrial fragmentation, whether and how Oma1 is involved in L-Opa1 processing and participates in the regulation of chemoresistance is unknown. Chemosensitive and chemoresistant ovarian (OVCA) and cervical (CECA) cancer cells were treated with cisplatin (CDDP). Mitochondrial dynamics and protein contents were assessed by immunofluorescence and Western blot, respectively. The requirements of Oma1 and p53 for CDDP-induced L-Opa1 processing, mitochondrial fragmentation, and apoptosis were examined by siRNA or cDNA. CDDP induces L-Opa1 processing and mitochondrial fragmentation in chemosensitive but not in chemoresistant cells. CDDP induced Oma1 40-kDa form increases in OV2008 cells, not in C13* cells. Oma1 knockdown inhibited L-Opa1 processing, mitochondrial fragmentation, and apoptosis. Silencing p53 expression attenuated the effects of CDDP in Oma1 (40 kDa) increase, L-Opa1 processing, mitochondrial fragmentation, and apoptosis in chemosensitive OVCA cells, whereas reconstitution of p53 in p53 mutant or null chemoresistant OVCA cells induced Oma1 (40 kDa) increase, L-Opa1 processing, mitochondrial fragmentation, and apoptosis irrespective of the presence of CDDP. Prohibitin 1 (Phb1) dissociates from Opa1-Phb1 complex and binds phosphorylated p53 (serine 15) in response to CDDP in chemosensitive but not chemoresistant CECA cells. These findings demonstrate that (a) p53 and Oma1 mediate L-Opa1 processing, (b) mitochondrial fragmentation is involved in CDDP-induced apoptosis in OVCA and CECA cells, and (c) dysregulated mitochondrial dynamics may in part be involved in the pathophysiology of CDDP resistance.     

Demonstration of exogenous nuclear histone H3 binding to mitochondria and subsequent cytochrome c release in cauliflower

The life cycle of a cell is partly regulated by the programmed cell death (PCD) process. From development to demise, a cell's PCD process must respond to external signals and internal factors mediated by mitochondria. Previous studies show that the release of histones into the cytosol caused by DNA damage or loss of nuclear integrity is correlated with apoptosis in mammalian cells. These released histones bind to mitochondria and permeabilize its inner and outer membranes, which causes the release of cytochrome c into the cytosol that leads to caspase activation and the demise of the cell. Owing to the high conservation of histones, we hypothesize that histone‐mediated cytochrome c release from mitochondria may be conserved across a wide range of eukaryotes. We investigated this histone–mitochondrial interaction in cauliflower using density‐gradient purified mitochondria and exogenous histones from a crude histone fraction, then added the exogenous histone fractions to the purified cauliflower mitochondria and analyzed the mitochondrial pellets and supernatants by immunoblotting against cytochrome c and H3. Our data clearly shows that histone‐enriched fractions elicited cytochrome c release from mitochondria, and that mitochondria bind exogenous histone H3.     

The morphological site of synthesis of cytochrome c in mammalian cells (Krebs cells)

In Krebs ascites-tumour cells, cytochrome c is segregated in the mitochondria and the level in microsomes could not be measured. At 22° in glucose–buffer Krebs cells synthesized a spectrum of proteins including cytochrome c. Mild osmotic shock in the presence of ribonuclease had little effect on incorporation of [¹⁴C]-leucine or [¹⁴C]valine into mixed mitochondrial protein but strongly inhibited synthesis of non-mitochondrial cytoplasmic proteins. Under these conditions, labelling of cytochrome c was also strongly inhibited. After pulse labelling of Krebs cells at 22° for 10min. the cytcchrome radioactivity found in mitochondria was higher than in microsomes. After addition of unlabelled amino acid as `chase' there was 137% increase in radioactivity of cytochrome c but only a 3% increase in radioactivity of whole-cell protein. It is concluded that the peptide chain of cytochome c is synthesized on cytoplasmic ribosomes. Mitochondria therefore do not have the character of self-replicating entities, but are formed by the cooperative function of messenger RNA of cytoplasmic ribosomes and, possibly, of intramitochondrial messenger derived from the mitochondrial DNA.     

Early septic shock induces loss of oxidative phosphorylation yield plasticity in liver mitochondria

We aimed to study the change in mitochondrial oxidative phosphorylation efficiency occurring at the early stage of septic shock in an experimental model. Thirty-six male Wistar rats were divided into two groups. In the first group, a cecal ligation and puncture (CLP) was carried out to induce septic shock for 5 h. The second group includes sham-operated rats and constitutes the control group. Blood gas analysis, alanine amino transferase, and lactic acid dosages were assayed 5 h after surgery. Liver mitochondria were isolated for in vitro functional characterization, including mitochondrial respiratory parameters, oxidative phosphorylation efficiency, oxi-radical production, membrane potential, and cytochrome c oxidase activity and content. Liver interleukin 1β (IL-1β) and tumor necrosis α mRNA levels were determined. Septic shock induced a severe hypotension occurring 180 min after CLP in association with a metabolic acidosis, an increase in plasma alanine amino transferase, liver IL-1β gene expression, and mitochondrial reactive oxygen species production. The rates of mitochondrial oxygen consumption and the activity and content of cytochrome c oxidase were significantly decreased while no alterations in the oxidative phosphorylation efficiency and inner membrane integrity were found. These results show that contrary to what was expected, liver mitochondria felt to adjust their oxidative phosphorylation efficiency in response to the decrease in the mitochondrial oxidative activity induced by CLP. This loss of mitochondrial bioenergetics plasticity might be related to mitochondrial oxidative stress and liver cytokines production.