The mitochondrial permeability transition pore (PTP) is a membrane protein complex assembled and opened in response to Ca2+ and oxidants such as peroxynitrite (ONOO–). Opening the PTP is mechanistically linked to the release of cytochrome c, which participates in downstream apoptotic signaling. However, the molecular basis of the synergistic interactions between oxidants and Ca2+ in promoting the PTP are poorly understood and are addressed in the present study. In isolated rat liver mitochondria, it was found that the timing of the exposure of the isolated rat liver mitochondria to Ca2+ was a critical factor in determining the impact of ONOO– on PTP. Specifically, addition of Ca2+ alone, or ONOO– and then Ca2+, elicited similar low levels of PTP opening, whereas ONOO– alone was ineffective. In contrast, addition of Ca2+ and then ONOO– induced extensive PTP opening and cytochrome c release. Interestingly, Cu/Zn-superoxide dismutase enhanced pore opening through a mechanism independent of its catalytic activity. These data are consistent with a model in which Ca2+ reveals a molecular target that is now reactive with ONOO–. As a test of this hypothesis, tyrosine nitration was determined in mitochondria exposed to ONOO– alone or to Ca2+ and then ONOO–, and mitochondrial membrane proteins were analyzed using proteomics. These studies suggest protein targets revealed by Ca2+ include dehydrogenases and CoA-containing enzymes. These data are discussed in the context of the role of mitochondria, Ca2+, and ONOO– in apoptotic signaling.
- permeability transition pore
peroxynitrite (ONOO–) is a biologically significant oxidant formed by the reaction of nitric oxide (NO·) with superoxide (2). Whereas the emerging paradigm is one of a beneficial antiapoptotic role for low physiological levels of NO·, it is generally accepted that ONOO– serves a proapoptotic role in many cell types (5). Despite significant evidence implicating ONOO– in apoptosis (20), its precise molecular mechanisms remain unclear, although the mitochondrion contains several protein targets that are particularly susceptible to ONOO–-dependent modification. These include components of the respiratory chain and manganese superoxide dismutase (SOD) (21).
An important mediator in the apoptotic cascade is the release of cytochrome c from the mitochondrion, leading to the assembly of the apoptosome and activation of downstream caspases (7). Cytochrome c release is regulated by the Bcl-2 family of proteins, and the target of these proteins in the cell is the mitochondrial permeability transition pore (PTP) (25). The molecular nature of the PTP and the precise mechanism by which it is linked to cytochrome c release (note that cytochrome c does not exit through the PTP) remain hotly debated. The key components of the PTP are believed to include the voltage-dependent anion channel, adenine nucleotide translocase, cyclophilin D, and a complement of Bcl/Bax family proteins.
Consistent with a mechanistic role for the PTP in cytochrome c release, several agents targeted at the molecular level of the PTP can either induce or inhibit cytochrome c release and apoptosis. Two examples are Ca2+ and ONOO–, which can act either alone or synergistically to induce PTP opening and cytochrome c release from isolated mitochondria (11). In a cellular setting, it is likely that the mitochondrion is simultaneously exposed to increased Ca2+, superoxide and, in some cases, NO. These are conditions in which it is predicted that ONOO– would be formed. Whereas it is recognized that oxidants and Ca2+ play a potentially important synergistic role in promoting mitochondrial dysfunction, the mechanisms that underlie such responses are unknown. Recently, it has been proposed that NO-dependent apoptosis in motor neurons can be enhanced by mutants of SOD (8, 9). Several mechanisms to explain these data have been advanced, and many involve the SOD-dependent posttranslational modification of mitochondrial proteins, including enhanced protein nitration. The effects of SOD on ONOO–-dependent PTP have not been examined. We hypothesized that the ONOO–-dependent posttranslational modification of mitochondrial proteins plays an important role and can be modified by exposure of the organelle to Ca2+. This concept was examined in isolated rat liver mitochondria using the measurement of PTP under a wide variety of conditions followed by a proteomics analysis. Herein, we report that Ca2+ exposes novel targets for tyrosine nitration within mitochondria.
Animals and reagents. Unless otherwise indicated, all reagents were of analytic grade from Sigma (St. Louis, MO). Peroxynitrite stock solutions (200–300 mM ONOO–) were prepared by reaction of acidified NaNO2 with H2O2 as previously described (4). Cu/Zn-SOD was inactivated by being boiled in H2O2 plus diethyldithiocarbamate for 1 h followed by dialysis. Mutant (D124N) SOD was a kind gift from Alvaro Estevez (Dept. of Physiology and Biophysics, University of Alabama at Birmingham). Animals were housed according to standard veterinary procedures under a 12:12-h light-dark photoperiod, with food and water available ad libitum. All animal procedures were performed with approval from the Institutional Animal Care and Use Committee.
Mitochondrial isolation. Liver mitochondria were isolated from male Sprague-Dawley rats (200–250 g body mass) by established differential centrifugation techniques (5) in isolation medium containing (in mM) 250 sucrose, 10 Tris, and 1 EGTA, pH 7.4 at 4°C. Care was taken to avoid the pale brown layer of light mitochondria and microsomes above the mitochondrial pellets. Final mitochondrial suspensions were kept on ice and used within 3 h of isolation.
Measurement of PTP opening. In isolated mitochondrial suspensions, mitochondrial PTP opening results in matrix swelling due to entry of water. The concurrent decrease in particulate light scattering of the suspension is assayed as a decrease in absorbance at 540 nm. Mitochondria (1 mg protein) were incubated in 1 ml buffer containing 25 mM sucrose, 95 mM mannitol, 40 mM HEPES, 5 mM succinate, and 10 μM rotenone, pH 7.2 at 37°C. Absorbance was monitored in a Gilford Response II spectrophotometer at 540 nm against a blank cuvet with buffer alone (5). Typical initial absorbances were ∼2 and within the linear range of the instrument. Additions to the cuvets are detailed in the figures and were made by pipetting stock solutions onto a plastic stirring paddle, followed by immersion and rapid rotation of the paddle to ensure efficient mixing and distribution.
Measurement of cytochrome c release. Mitochondrial swelling was terminated by the addition of 2 mM EGTA and 0.5 μM cyclosporin A (Calbiochem; San Diego, CA) to the cuvet, followed by centrifugation (15,000 g, 10 min) at 4°C. Pellets were frozen for proteomics analysis (see Mitochondrial proteomics). Twenty microliters of supernatant were then mixed with 20 μl of SDS-PAGE loading buffer and separated on 18% polyacrylamide gels. Gels were electroblotted to nitrocellulose membranes and probed with a mouse monoclonal antibody to denatured cytochrome c (BD Pharmingen; San Diego CA), followed by horseradish peroxidase (HRP)-linked anti-mouse secondary antibody and enhanced chemiluminescence detection (AP Biotech; Piscataway, NJ). Bands were quantified by densitometry using “Scion Image” software (www.scioncorp.com).
Mitochondrial proteomics. A significant obstacle that has delayed the study of proteins in mitochondria is the difficulty in applying a proteomics approach. This is because traditional two-dimensional gel techniques are poorly adapted for the analysis of membrane proteins (6). Specifically, the hydrophobic proteins of the mitochondrial inner membrane precipitate at the basic pole during isoelectric focusing. We therefore used two-dimensional blue-native gels (BN-PAGE) coupled to analysis of protein modifications by Western blotting and subsequent protein identification by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry to study tyrosine nitration in mitochondrial membrane proteins (6, 22). In this technique, the complexes of the respiratory chain are separated in a native enzymatically intact form in the first dimension. This is achieved by mild detergent solubilization, and the dye Coomassie blue is used to stabilize the complexes and confer a negative charge, facilitating electrophoresis. Lanes are then cut from the gel, turned through 90°, and separated in the second dimension by denaturing Tris-Tricine electrophoresis, resulting in a column of vertically aligned subunit protein spots for each of the mitochondrial membrane complexes. The full methodology for this type of gel is detailed in Ref. 6.
Detection of protein modifications by Western blotting. All two-dimensional gels were run in duplicate. One gel was stained with Coomassie blue and the other electroblotted to nitrocellulose. Blots were probed with rabbit polyclonal antibodies to 3-nitrotyrosine (Upstate Biochem; Waltham, MA). Detection was by HRP-linked secondary antibodies and ECL, as detailed above.
Identification of proteins. Gels swell during staining and destaining, relative to their corresponding nitrocellulose blots. Therefore, developed blots were electronically resized to match the gels. Blots and gels were then aligned on a light box, and spots corresponding to modified proteins were excised for identification. Processing, trypsinization, and MALDI-TOF peptide mass fingerprinting were performed in University of Alabama's proteomics core facility (www.uab.edu/proteomics) as previously described (6).
In this investigation, the differential effects of ONOO– and Ca2+ on the mitochondrial PTP opening were examined. In these experiments, concentrations of Ca2+ were selected that were submaximal for the PTP so that the potential for ONOO– to promote pore opening could be assessed. Figure 1 shows typical swelling traces following exposure of isolated mitochondria to Ca2+ and/or ONOO–. The swelling is inhibited by cyclosporin A, indicating that this phenomenon is indeed due to PTP opening. Figure 1A, inset, shows the effects of ONOO– exposure after Ca2+ on cytochrome c release; these data parallel the swelling data. Figure 1B shows a concentration response to ONOO–, indicating that swelling reaches a maximum at around 200 μM bolus ONOO–. There are several parameters of the swelling curve that could be used for quantitation, including maximal swelling amplitude, maximum or average swelling velocity, lag time, or derivatives thereof. Two such measurements are shown in Fig. 1B and correlate well with each other (inset), indicating that either of these parameters can be used to quantify mitochondrial swelling data.
Figure 2 shows that the time of ONOO– addition to mitochondria, relative to that of Ca2+, affects the extent of PTP opening. Addition of ONOO– before Ca2+ (Fig. 2A, trace A) does not alone trigger PTP opening and only slightly increases the degree of swelling versus Ca2+ alone (control trace). However, addition of ONOO– immediately after Ca2+ results in greatly enhanced PTP opening (Fig. 2A, trace B). Interestingly, a longer incubation time between Ca2+ and ONOO– addition appears to diminish the degree of swelling. This suggests that the ability of Ca2+ to modulate ONOO– opening of the PTP may be transient or reversible. These data are quantified in Fig. 2B, in which both the extent of swelling (solid bars) and rate (open bars) were determined. It is evident that the degree of PTP opening/swelling is more sensitive to ONOO–-Ca2+ than is the maximal rate of swelling, which reaches the same level irrespective of the time of ONOO– addition following Ca2+.
The role of nitrotyrosine (N-Tyr) in the processes leading to apoptosis is a subject of debate because it is the mechanism by which N-Tyr is formed in vivo (26, 27). It has been proposed that under certain conditions, Cu/Zn-SOD1 is able to catalyze nitration by ONOO– (3, 15). Therefore, we sought to test the ability of SOD1 to enhance the effects of ONOO– on mitochondrial PTP. Figure 3 shows that addition of SOD1 before ONOO– or Ca2+ causes an increase in the rate of swelling, consistent with a possible involvement of N-Tyr formation in the mechanism of ONOO–-induced PTP opening. Figure 4 shows the effects of SOD1 on the ONOO– dose-response curve (cf. Fig. 1) and suggests that SOD1 effectively lowers the threshold for ONOO– induction of PTP opening.
Figure 5 shows a time course study in which SOD1 was added at different times relative to Ca2+ and ONOO–. Addition of SOD1 at any time before ONOO– results in increased PTP opening (relative to no SOD1 addition), whereas addition of SOD1 after ONOO– had no effect. Furthermore, Fig. 5B shows that the effects of SOD1 are not due to formation of H2O2. This is an important control experiment, because oxidants such as t-Bu-OOH have previously been shown to induce PTP opening (28). Addition of catalase to remove H2O2 did not significantly diminish the effects of SOD, and addition of H2O2 alone did not affect the PTP opening. Interestingly, a mutant form of SOD1 that has been implicated in enhanced N-Tyr formation and toxicity in amyotrophic lateral sclerosis (ALS) (8, 23) also enhanced Ca2+ plus ONOO–-induced PTP opening by a similar degree to wild-type SOD1. A bacterial MnSOD (SOD2), which does not catalyze tyrosine nitration, did not affect PTP opening. It is not clear whether the enzymatic activity of SOD1 is important for this phenomenon, because the method for inactivation of SOD1 (boiling in H2O2) may also remove other nonenzymatic properties of the enzyme, such as tertiary structure. Addition of authentic 3-N-Tyr, nitrated SOD1, or nitrated BSA did not induce PTP opening (result not shown), suggesting that if N-Tyr formation is involved in the mechanism of the PTP, it is at the level of mitochondrial protein nitration. Furthermore, as shown in Fig. 6, SOD1 did not affect PTP opening induced by Ca2+ alone or by Ca2+ plus phosphate, suggesting a unique interaction with ONOO– rather than a nonspecific effect on PTP opening in general.
One of the most widely characterized effects of ONOO– on proteins is the nitration of tyrosine residues. Figure 7 shows N-Tyr Western blot data for mitochondria treated with ONOO– or Ca2+. Figure 7A indicates three classes of proteins: 1) endogenously nitrated, 2) nitrated by ONOO– alone, and 3) nitrated more in the presence of Ca2+. To identify some of the proteins nitrated by ONOO–, mitochondria were separated on two-dimensional BN gels, and a typical gel is shown in Fig. 7B. Gels were Western blotted against N-Tyr, and typical blot results are shown in Fig. 7C. Similar to Fig. 7A, these blots show endogenously nitrated proteins plus proteins nitrated either by ONOO– alone or by Ca2+ plus ONOO–. With the blots and gels aligned, spots were then excised and identified by mass spectrometry (Table 1). Table 1 shows a list of nitrated proteins obtained from several experiments. Notably, dehydrogenases and CoA-containing enzymes appear to be major targets for ONOO–-dependent nitration.
Another interesting observation made from the two-dimensional gels was the loss of a major spot following Ca2+ treatment. Figure 8 shows this in more detail, indicating that the protein running at a high molecular weight in the first dimension, between complexes III and IV, is diminished in magnitude on Ca2+ exposure. Mass spectrometric analysis revealed this spot to be glutamate dehydrogenase (GDH). Furthermore, GDH is one of the enzymes identified as being more nitrated by ONOO– after Ca2+ exposure (Table 1). Therefore, it is interesting to speculate that by changing the three-dimensional structure and multimerization of GDH, Ca2+ has exposed a target on the enzyme for nitration.
It has been demonstrated in a number of studies that reactive nitrogen species can exhibit different effects on mitochondrial function and cells, including the propensity to initiate apoptosis (9, 20). NO at low physiological concentrations can inhibit apoptosis in response to a number of stimuli and in isolated mitochondria prevents both the permeability transition and cytochrome c release (5). In contrast, ONOO– is both proapoptotic in cells and enhances the PTP in isolated mitochondria (9, 20). It is now thought that one of the main reactions through which ONOO– can modify protein function is through nitration, which can be enhanced by SOD1 but not SOD2 (3, 15). This reaction is postulated to make an important contribution to the death of motor neurons in ALS (8, 23). Interestingly, it has recently been suggested that a mitochondrial pool of Cu/Zn-SOD may be the chief mediator of this pathology (14). The effects of Ca2+ on isolated liver mitochondria are many and varied, but recently it was reported that high concentrations of Ca2+ can cause rearrangement of cardiolipin and lipid rafts in the mitochondrial membranes, which may facilitate PTP opening (12, 28). However, the mechanisms through which ONOO– in the presence of absence of SOD1 enhances the mitochondrial PTP are not understood in detail and are the subject of the present study.
In the first series of experiments, we demonstrate that the timing of exposure of ONOO– and Ca2+ required to elicit PTP in isolated mitochondria is critical. Because ONOO– must be added after Ca2+ to enhance swelling, it suggests that the reactive nitrogen species (RNS) alone is not sufficient to promote a proapoptotic response in the isolated organelle. This effect is evident using several parameters to assess the extent of pore opening, including cytochrome c release. As noted in previous studies, the ONOO–-dependent promotion of the PTP is cyclosporin sensitive, consistent with the “classical” PTP in isolated mitochondria (20). The lowest concentration of ONOO– required to elicit a significant effect on pore opening is ∼50 μM. It should be noted that under these conditions, the effective concentration of ONOO– capable of reaching the mitochondria is estimated to be in the low micromolar range because of the intrinsic instability of the compound at physiological pH and competing reactions with components of the mitochondrial incubation buffer. In addition, these experiments are conducted at a high concentration of mitochondria compared with those found in the cell due to the limitations of the biochemical models used to assess the PTP. Given these constraints, it is then likely that low nanomolar concentrations of ONOO– produced at or near the mitochondrion within the cell can lead to the responses described here. It is clear that a reaction product between ONOO– and a mitochondrial component is required to promote PTP opening, because neither decomposed ONOO– (not shown), or ONOO– added to the buffer before mitochondria (essentially the same as the decomposed ONOO– control) had any effect on PTP opening.
It is thought that SOD1 can promote nitrative reactions mediated by ONOO–, and we therefore sought to determine the effects of this enzyme on ONOO–-induced PTP opening. Interestingly, SOD1, but not SOD2 or inactive SOD, enhanced the ONOO–-induced PTP opening. Addition of SOD after ONOO– did not enhance PTP opening, suggesting that SOD1 is interacting with ONOO– and not with Ca2+ or with the decomposition products of ONOO– (because the half-life of ONOO– is <1 s in this system). The product of SOD catalysis H2O2 had no effect on the PTP opening, but a mutant form of SOD that is competent in mediating nitration but with much reduced SOD activity did enhance the PTP, suggesting an interactive mechanism between SOD1 and ONOO–. Interestingly, the controversial issue of whether a subset of SOD1 exists in the mitochondrial intermembrane space has recently been reawakened (19, 29). Such a location for SOD1 may offer a unique ability for ONOO–-mediated nitration of proteins in the intermembrane space, including those involved in assembly of the PTP.
To investigate which proteins are nitrated by ONOO–, we used two-dimensional BN gels and N-Tyr Western blotting. This type of gel has significant advantages over the traditional isoelectric focusing SDS-PAGE two-dimensional gels, offering enhanced representation and resolution of membrane proteins such as those of the mitochondrial respiratory chain (6). The data in Fig. 7 and Table 1 indicate that several proteins are endogenously nitrated in mitochondria. In addition, there are proteins nitrated by ONOO– alone and further nitrated in response to Ca2+ plus ONOO–. One protein, GDH, was identified as a potential target revealed by Ca2+ exposure to the organelle and may also be linked to the mechanisms of oxidant-induced PTP.
In the signaling cascade of apoptosis, the activation of poly-ADP-ribose polymerase (PARP) is an important event, leading to depletion of cytosolic NADH levels due to the use of this cofactor in an enzymatic activity that can ribosylate cysteines residues on proteins (18). Notably, PARP is activated by ONOO– (24). In addition, ADP ribosylation of mitochondrial proteins has been reported (10) and is thought to be mediated by mitochondrial congener of PARP known as mitochondrial ADP-ribosyl transferase (MART) (16). One of the proteins identified as a target of MART is GDH, whereupon ADP ribosylation leads to dissociation of the GDH hexamer into monomers (13). Such a dissociation of GDH is consistent with our data in Fig. 8, showing the loss of a GDH band from the first dimension of the BN gel at ∼300 kDa, following treatment of mitochondria with Ca2+. Significantly, mitochondrial ADP-ribosylcysteine hydrolase, the enzyme that reverses the ADP ribosylation, is activated by Mg2+ (13), although it is unknown whether Mg2+ can perform a similar enhancement of ONOO–-induced PTP opening as reported here for Ca2+. In addition, a change in the tertiary structure of GDH would be postulated to expose new residues on the surface of the protein, possibly as a target for tyrosine nitration. This is in agreement with the data in Fig. 7 showing that GDH is more nitrated following Ca2+ exposure. Whereas the precise role of GDH nitration/dissociation, if any, in PTP opening is unknown, it is interesting to speculate that in whole cells, loss of this important enzyme may have profound implications for mitochondrial metabolism and ATP generation and thus for the execution of the apoptotic cascade.
Another possibility is that the ability of Ca2+ to enhance ONOO–-induced PTP opening is independent of any effects on tyrosine nitration. For example, several other protein modifications such as S-nitrosation, S-nitration, oxidation, or cross-linking can be mediated by ONOO–. Indeed, we have shown using novel mitochondrial thiol-labeling compounds that ONOO– is a potent effector of thiol modifications within the mitochondrial matrix (17). Alternatively, Ca2+ may be acting independent of exposure of protein targets. For example, the activation of MART would be expected to deplete the mitochondrial NADH pool, which has been shown to be a potent modulator of PTP opening. Ca2+ may also affect the mitochondrial glutathione pool, thereby changing the susceptibility of mitochondrial proteins to ONOO– via removal of this important antioxidant. Interestingly, our results agree well with those of Aulak et al. (1), showing that certain classes of protein appear to be preferentially nitrated in response to RNS. It is tempting to speculate that these proteins (in this case, CoA-metabolizing enzymes and dehydrogenases) may contain a specific motif that enhances susceptibility to nitration.
In summary, it appears that the interplay between ONOO– and the PTP is complex, with several factors possibly involved in the execution of PTP and eventually apoptosis. These include Ca2+, tyrosine nitration, protein tertiary structure, ADP ribosylation, and mitochondrial antioxidant status. These studies indicate a possible molecular basis for the enhancement of RNS-dependent cytotoxicity under conditions of stress in which cytosolic calcium is elevated. These findings have potential implications for the pathogenic mechanisms that underlie neurodegenerative disease and chronic inflammation.
The authors are grateful to Anita Pinner for technical assistance and Professors Joseph Beckman (Oregon State University) and Alvero Estevez (University of Alabama at Birmingham) for stimulating discussions.
Current address of P. S. Brookes: Dept. of Anesthesiology, Univ. of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642 (E-mail:).
This study was supported by National Institutes of Health Grant AA-13395 (to V. M. Darley-Usmar).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2004 by the American Physiological Society