Xanthurenic acid translocates proapoptotic Bcl-2 family proteins into mitochondria and impairs mitochondrial function

Background Xanthurenic acid is an endogenous molecule produced by tryptophan degradation, produced in the cytoplasm and mitochondria. Its accumulation can be observed in aging-related diseases, e.g. senile cataract and infectious disease. We previously reported that xanthurenic acid provokes apoptosis, and now present a study of the response of mitochondria to xanthurenic acid. Results Xanthurenic acid at 10 or 20 μM in culture media of human aortic smooth muscle cells induces translocation of the proteins Bax, Bak, Bclxs, and Bad into mitochondria. In 20 μM xanthurenic acid, Bax is also translocated to the nucleus. In isolated mitochondria xanthurenic acid leads to Bax and Bclxs oligomerization, accumulation of Ca2+, and increased oxygen consumption. Conclusion Xanthurenic acid interacts directly with Bcl-2 family proteins, inducing mitochondrial pathways of apoptosis and impairing mitochondrial functions.


Background
Xanthurenic acid is an endogenous molecule derived from tryptophan degradation by indoleamine-2, 3-dioxygenase. Indoleamine-2, 3-dioxygenase is induced in infectious diseases by liposacharides, interferon-α, and superoxide radicals [1]. Xanthurenic acid is enzymatically formed by transamination of 3-hydroxykynurenine by kynurenine-aminotransferase [2]. Kynurenine-aminotransferase, has been cloned from mitochondria and cytoplasm [3,4]. Xanthurenic acid accumulates with age in senile cataract [5][6][7]. Previously, we reported that this acid leads to apoptotic-like cell death [8][9][10], its BH3 domain in the cytoplasm being responsible for targeting the protein to mitochondria, resulting in cytochrome c release [11,12]. We now report that proteins with a BH3-domain (Bax, Bak, Bcl-x s , Bad) are transformed in the presence of xanthurenic acid to their proapoptotic-inducing state and translocated to mitochondria. In vitro, xanthurenic acid leads to covalent oligomerization of Bax, and Bcl-x s . It also alters mitochondrial Ca 2+ transport, and increases oxygen consumption.

Xanthurenic acid provokes translocation of Bak, Bax, Bclx s , and Bad to mitochondria
The fate of Bax, Bak, Bclx s , and Bad in cultures of aortic human smooth muscle cells (HSMC) has been compared with control cells, using the antibody against Bax, which stains cells uniformly (Fig. 1A). 10 µM xanthurenic acid results in Bax being translocated to mitochondria (Fig.  1B), and at 20 µM, Bax seems to be translocated from mitochondria to nuclei (Fig. 1C). However, at the latter concentration, some Bax remained detectable in mitochondria (Fig. 1C). Granular bodies strongly staining with anti-Bax antibodies (green) were detected in the nucleus (Fig. 1C). Translocation of Bax to the nucleus during apoptosis has previously been reported [13]. Proapoptotic proteins Bak, Bclx s and Bad ( Fig. 2A,2B,2C) at 10 and 20 µM xanthurenic acid (Fig. 2D,2E,2F) were translocated into mitochondria.

Xanthurenic acid leads to oligomerization of cytoplasmic Bcl-x s and Bax in the rat liver mitochondrial fraction
Both Bcl-x s and Bax were found in the 10,000 g mitochondrial fraction of rat liver. When it was incubated with xanthurenic acid at 5-10 µM (1 mg of protein/ ml), cleavage of Bax with formation of p18 was observed. Both anti-Bax and anti-Bcl-x s recognized the N-terminal part of the proteins, suggesting that the cleavage was more towards the C-terminal domain. When the concentration of xanthurenic acid was increased to 20-40 µM (1 mg protein/ ml), Bax dimerized (Fig. 3A). Western blot analysis showed an increase of staining and dimerization of Bcl-x s in the presence of xanthurenic acid (Fig. 3B). In the mitochondrial fraction, incubation of Bcl-x s and Bax, with xanthurenic acid led to stronger attachment of the correspondent IgG antibody than in control cells, indicating some form of posttranscriptional modification. Dimers of the proapoptotic proteins in the presence of xanthurenic acid showed up on SDS-PAGE, confirming the formation of covalent crosslinking. Bax and Bclx s were faintly detectable in the mitochondrial pellets. Cyto-chrome c release was observed in the same protein extract (Fig. 3C).

Mitochondrial Ca 2+ release
Ca 2+ release was measured with Arsenazo III at a wavelength of 685/675 nm, using an Aminco DW-2 spectrophotometer. Xanthurenic acid at 15 µM (1 mg protein/ ml) did not alter the release of Ca 2+ , but inhibition did occur at 66 µM (1 mg protein/ml). Ca 2+ retained in mitochondria at this concentration was liberated after addition of 1 µM carbonyl cyanide-m-chlorophenylhydrazone (CCCP), which is known to provoke a loss of potential across mitochondrial membranes (Fig. 4). These results indicate that xanthurenic acid is involved in the modification of mitochondrial Ca 2+ release, but does not lead to a loss of the mitochondrial membrane potential.

Consumption of O 2 by mitochondria in the presence of xanthurenic acid
Consumption of O 2 was measured with a Clarke-type electrode (Fig. 5). Mitochondrial respiration was supported with 2.5 mM potassium succinate prior to xanthurenic acid being added at 0.4 (Fig. 5, curve 1) or 4 µM (Fig. 5, curve 2). Xanthurenic acid stimulated oxygen consumption in a concentration-dependent manner.

Discussion
Apoptosis in the presence of xanthurenic acid is associated with cytochrome c release [8][9][10]. Our investigations show that xanthurenic acid induces the translocation of proapoptotic Bcl-2 proteins to mitochondria. A posttranscriptional modification of these proteins is suggested by the Primary cell cultures human smooth muscle cells direct crosslinking of Bax and Bcl-x s in isolated mitochondria. Previously, BID was reported to change the conformation of Bax [14]. Recently, we reported that accumulation of xanthurenic acid in cells interrupts the interaction of Bad with signalling proteins of the 14-3-3 family [10]. However, here we have shown that Bax is dimerized in isolated mitochondria, indicating that xanthurenic acid itself induces a conformational change of the proteins. Using Western blotting, Bax in mitochondria incubated with xanthurenic acid was detected more readily than in controls. This phenomenon was previously reported to indicate a change in Bax folding which results in a better exposition of the N-terminal part of Bax for the antibody after exposure to apoptotic stimuli [15]. Furthermore, xanthurenic acid appeared to modify the folding of proteins [16], which could be the reason for the increased IgG binding to proteins in the presence of xanthurenic acid.
Our results indicate that xanthurenic acid is an endogenous crosslinker of Bax and Bclx s proteins with a BH3 domain. Xanthurenic acid leads to translocation of BH3 proteins into mitochondria in human smooth muscle aortic cells. However, in isolated mitochondria, dimerized Bax and Bclx s found in the mitochondrial supernatant, are almost undetectable in the pellets, indicating that an additional cytoplasmic protein is necessary for translocation of these proteins into mitochondrial membranes. Therefore, the observed cytochrome c release in the presence of xanthurenic acid in isolated mitochondria is not mediated by Bax dimerization. At lower concentrations of xanthurenic acid, cleavage of Bax produced p18, which has been previously shown to enhance apoptosis [17]. Bax dimerization also has been shown to induce caspaseindependent cell death [18]. Xanthurenic acid at 20 µM causes Bax translocation into the nucleus, and plays a key role in apoptosis, because Bax/Bak deficient cells were Primary cell cultures human smooth muscle cells resistant to the apoptotic stimuli staurosporine and etoposide [19]. The fact that xanthurenic acid can directly provoke Bax and Bcl-x s crosslinking suggests that an accumulation of xanthurenic acid in a cell is sufficient to induce the cell death. Xanthurenic acid might be oxidized in vivo to its semiquinone, and further to quinone-di-oxoxanthurenic acid, as previously described [6]. The semiquinone is an oxidative stress [20], which can lead to uncoupling of mitochondria, characterized by an increase of O 2 consumption; in keeping, we noted an increased consumption of O 2 in the presence of xanthurenic acid. Previously it was reported that Bax is only toxic in the presence of active oxidative phosphorylation [21]. Our results indicate that at 20 µM xanthurenic acid, Bax is translocated into the nucleus and Ca 2+ release from mito-chondria is blocked. Xanthurenic acid accumulation in mitochondria does not affect mitochondrial membrane potential, because Ca 2+ was accumulated in the mitochondria and CCCP was able to release Ca 2+ under these conditions. Previously it was reported that Bax/Bax-induced apoptosis was associated with an uptake of mitochondrial Ca 2+ by an unknown mechanism. An excessive Ca 2+ load may induce apoptosis by stimulating the release of apoptosis-promoting factors from the mitochondrial membrane space to the cytoplasm, leading to impaired mitochondrial function (for review [22]).
The results presented here indicate that the accumulation of xanthurenic acid in the human aortic smooth muscle cell causes a proapototic conformation of the mitochondrial proteins, Bax, Bak, Bcl-x s , and Bad. It was previously reported, that mitochondrial Bax plays an essential role in cytochrome c release, which can occur via Ca 2+ -dependent and -independent mechanisms [23]. Bax-driven apoptosis may be important for development of diabetes, atherosclerosis and neurological disorder [24][25][26][27][28]. We surmise that an accumulation of the endogenous aromatic amine, xanthurenic acid, may lead to organ failure.

Reagents
The following polyclonal antibodies were obtained from Santa Cruz Biotechnology Inc. CA, USA: antibody against cytochrome c, against N-terminal part of Bax, Bak, Bcl-x s , and Bad. IgG secondary antibodies-Texas Red conjugated were from Santa Cruz or secondary antibodies IgG-Alexa 488 conjugated Molecular Probes (Leiden, The Netherlands). Carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and Arsenazo III were from Sigma.

Preparation of human smooth muscle cells primary culture
The normal human aortic smooth muscle cells were from PromoCell GmbH, Heidelberg, Germany.

Western blot analysis of protein from rat liver mitochondrial fraction
Rat liver mitochondria (80-90 mg protein per ml) were isolated, as described previously [29]. The 10,000 g mitochondrial fraction of cells was used (referred herein as mitochondria). The mitochondria were in MSH buffer Western blot analysis of supernatant from rat liver mito-chondria incubated for 1 h in the presence of xanthurenic acid for Bax, Bcl-x s and cytochrome c (210 mM mannitol, 70 mM sucrose, 5 mM Hepes, pH 7.35 (4°C, 2 M KOH). Mitochondria (~3 mg protein per 100 µl of reaction mixture) were incubated for 1 h at room temperature without xanthurenic acid or in the presence of the increasing concentrations of xanthurenic acid from 5 to 40 µM per mg protein in the presence of the following protease inhibitors: 0.1 mM phenyl-methylsulfonyl fluoride, and leupeptin, aprotinin, and pepstatin, each at 1 µg/ ml. Thereafter, the sample was centrifuged for 10 min at 14,000 g, and the supernatant boiled in loading-buffer for 5 min. Proteins at 100 µg per lane were separated by SDS-PAGE containing 12.5% acrylamide. After transfer to Hybond ECL membrane (Amersham Pharmacia Biotech AB, Uppsala, Sweden), the proteins were probed with the appropriate antibodies. Chemiluminescence ECL system (Amersham Pharmacia Biotech AB, Uppsala, Sweden) was used for the detection of peroxidase-conjugated secondary antibody.

Immunofluorescence studies
Cells grown on glass coverslips were fixed for 10 min at room temperature in 4% paraformaldehyde in 0.1 M PIPES, pH 6.8, washed in PBS and permeabilized for 5 min in PIPES containing 0.05% saponin (65 µl per coverslip), washed in PBS, incubated for 10 min in cold acetone for additional fixing and permeabilisation, and again washed in PBS. Cells were incubated for 1.5 h with the first antibody diluted in PBS containing 1% bovine serum albumin, and after further washing incubated for 1.

Measurement of calcium uptake and release and O 2 consumption in liver mitochondria in the presence of xanthurenic acid
An Aminco DW-2A spectrophotometer was used at 685-675 nm the presence of 50 µM Arzenazo III, and mitochondrial respiration was measured using a Clarkeelectrode as described previously [29]. Briefly, mitochondria (1 mg of mitochondrial protein/ml) were energized with 2.5 mM potassium succinate in the presence of rotenone. After Ca 2+ uptake was complete, the uptake blocker, ruthenium red (RR), was added to see net release Ca 2+ in the presence or absence of xanthurenic acid.
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