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Ca2+-induced release of mitochondrial m-calpain from outer membrane with binding of calpain small subunit and Grp75
Archives of Biochemistry and Biophysics 507 (2011) 254–261
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Ca2+-induced release of mitochondrial m-calpain from outer membrane with binding of calpain small subunit and Grp75 Taku Ozaki a, Tetsuro Yamashita b, Sei-ichi Ishiguro a,⇑ a b
Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan Department of Food Science and Biochemistry, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
a r t i c l e
i n f o
Article history: Received 21 October 2010 and in revised form 30 November 2010 Available online 9 December 2010 Keywords: Calpain Calpain small subunit Grp75 Activation Mitochondrial outer membrane Apoptosis
a b s t r a c t Although mitochondrial l- and m-calpains play significant roles in apoptotic cell death, their activating mechanisms have not been determined. The purpose of this study was to determine the core factors that are involved in activating mitochondrial outer membrane (OM)-bound calpains. To accomplish this, we solubilized OM-bound calpains and separated them by DEAE-Sepharose column chromatography, and identified them by immunoblots. We also determined the core factors that activated the OM-bound calpains and release them from the OM by calpain assays, immunoprecipitations, and immunoblots. The OM-bound m-calpain large subunit was not associated with the small subunit or with Grp75 chaperone. Free calpain small subunit was located in the IMS and caused the release of the OM-bound m-calpain large subunit from the OM together with Grp75, ATP, and Ca2+. Our results showed that the activating mechanism of mitochondrial OM-bound m-calpain and the release of mitochondrial m-calpain from the OM have important implications in facilitating apoptotic cell death. Published by Elsevier Inc.
Introduction Calpains are Ca2+-activated neutral cysteine proteases that modulate the functions of many specific substrates by limited proteolysis [1]. In humans, 14 calpain isoforms have been identified as the catalytic large subunit and two isoforms for regulatory small subunits [1,2]. Among them, l-calpain and m-calpain large subunits, calpain small subunit, and calpain 10 exist in mitochondria [3]. The mitochondrial calpains play significant roles in pathophysiological conditions including apoptotic and necrotic cell deaths [3]. Endo et al. showed that mitochondrial calpains inactivate mitochondrial aspartate aminotransferase in the rat retina under ischemic and hypoxic conditions [4]. We found that mitochondrial l-calpain modulates apoptotic cell death by limiting the cleavage of the apoptosis-inducing factor (AIF)1 [5,6]. We also demonstrated that mitochondrial m-calpain plays a significant role in the
⇑ Corresponding author. Address: Division of Cell Technology, Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan. Fax: +81 172 39 3780. E-mail address: [email protected] (S.-i. Ishiguro). 1 Abbreviations used: AIF, apoptosis-inducing factor; tAIF, truncated AIF; VDAC, voltage-dependent anion channel; Grp75, glucose-regulated protein 75; GAPDH, glyceraldehyde phosphate dehydrogenase; OM, outer membrane; IMS, intermembrane space; IM, inner membrane; ATP, adenosine 50 -triphosphate; TTP, thymidine 50 triphosphate; GTP, guanosine 50 -triphosphate; CTP, cytidine 50 -triphosphate. 0003-9861/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.abb.2010.12.003
release of truncated AIF from mitochondria through VDAC–Bax complex [7]. Very recently, we proved that the mitochondrial calpains induce photoreceptor apoptosis in the Royal College of Surgeon’s (RCS) rats, which are widely employed as an animal model of human retinitis pigmentosa [8]. Although mitochondrial calpains are involved in a variety of pathological conditions [8–11], how they are activated has not been determined. Earlier, we found that the mitochondrial mcalpain large subunit was located mainly in the OM [7], but the calpain activity in the OM was very weak [5]. These observations suggested that mitochondrial m-calpain in the OM is regulated by not only Ca2+ but also other factors such as calpain activators, inhibitors, and molecular chaperones. The purpose of this study was to determine the core factors that are involved in activating OM-bound calpains. We have shown that mitochondrial l- and m-calpains are associated with the molecular chaperones such as ERp57 and Grp75, respectively, in the mitochondrial intermembrane space (IMS) [6,7]. In addition, we detected three types of calpain small subunits in the IMS using non-denaturing gels: the first was a mitochondrial l-calpain large subunit-associated calpain small subunit, the second was a mitochondrial m-calpain-associated calpain small subunit, and the third was an unknown calpain large subunit-associated or free calpain small subunit [6]. To identify the activating factors of OM-bound calpains, we determined what kinds of calpain isoforms were present in the OM and investigated the effects of Ca2+, ERp57, Grp75, and calpain small subunit.
T. Ozaki et al. / Archives of Biochemistry and Biophysics 507 (2011) 254–261
Materials and methods Materials The detergents used to solubilize the mitochondrial outer membrane (OM) were: sodium deoxycholate (Fluka, Milwaukee, WI), Nonidet P-40 and Octyl-glucoside (Sigma Aldrich Corp., St. Louis, MO), digitonin, CHAPS, n-dodecyl-b-D-maltoside, Triton X-100, and Tween 20 (Wako). Grp75 protein was purchased from Abcam (Cambridge, MA). Adenosine 50 -triphosphate (ATP), thymidine 50 triphosphate (TTP), guanosine 50 -triphosphate (GTP) and cytidine 50 -triphosphate (CTP) were purchased from Sigma. The antibodies purchased were: rabbit polyclonal antibodies against l-calpain large subunit domain IV (Sigma); anti-m-calpain large subunit domain III (Abcam); anti-calpain small subunit domain V (Abcam); anti-Grp75 (Santa Cruz Biotechnology Inc., CA); anti-adenylate kinase 2 (Santa Cruz); anti-GAPDH (Santa Cruz); anti-VDAC (Calbiochem); anti-pyruvate dehydrogenase (Molecular Probes, Eugene, OR); mouse monoclonal anti-trans-Golgi network38 (Abcam); rat monoclonal anti-ZO-1 (Chemicon, Temecula, CA); and normal rabbit IgG (Santa cruz). Rabbit anti-ERp57 antiserum was prepared against the 17-amino acid C-terminal peptide (VIQEEKPKKKKKAQEDL) of human ERp57 as described [12]. Subfractionation of rat liver mitochondrial outer membrane and intermembrane space All of the procedures were carried out at 4 °C, following the method of Parson et al. [13]. The mitochondrial fraction obtained as described [5] was resuspended in two volumes of 20 mM potassium phosphate buffer, pH 7.4, and allowed to stand at 4 °C for 1 h. The resuspended sample was centrifuged at 3000g for 10 min. The supernatant was centrifuged at 105,000g for 30 min, and the pellet was used as the OM fraction, and the supernatant was used as the IMS fraction. The purity of the mitochondrial OM and IMS was determined by immunoblot analysis with the following antibodies; anti-VDAC antibody for mitochondrial OM, antiadenylate kinase 2 (AK2) for intermembrane space (IMS), antiAIF antibody for inner membrane (IM), pyruvate dehydrogenase (PDH) antibody for matrix, anti-GAPDH antibody for cytosolic fraction, anti-ZO-1 for plasma membrane, anti-calnexin for ER, antitrans-Golgi network-38 for Golgi apparatus as shown (see Ref. [7]). High purities of the mitochondrial OM and IMS were obtained.
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Unbound proteins were eluted with 770 ml of buffer B. The bound proteins were eluted with a linear gradient of 50–300 mM NaCl in buffer A in a total volume of 1.0 L. Four calpain-like activity peaks were detected. The fractions containing calpain-like activity were collected and concentrated by Amicon PM-10 membrane (Millipore Co., Bedford, MA). The concentrates were used for Western blot analyses with anti-l-calpain and anti-m-calpain large subunits, anti-calpain small subunit, anti-ERp57 and antiGrp75 (Fig. 2B). They were also used for immunoprecipitation with anti-l-calpain large subunit, anti-calpain small subunit, and antiERp57 (Fig. 2C). The protein concentration was measured by the Bradford et al. method with bovine serum albumin (BSA) as the standard [14]. We also determined whether that the proteins in the mitochondrial intermembrane space (IMS) were involved in the activation of mitochondrial OM proteins. Fifty-five micrograms of IMS proteins were added to 50 ll of each fraction and incubated at 4 °C for 2 h. Then the calpain activity of each sample was measured (Fig. 3D). Column chromatography of mitochondrial IMS-localized
l- and m-calpains Rat liver mitochondrial IMS were prepared from 30 SD rats (8 weeks-old) as described above. Partial purification of mitochondrial l- and m-calpains was accomplished by DEAE-Sepharose CL-6B column chromatography as we described [5–7] (Fig. 2A). The prepared IMS fraction was dialyzed overnight against 2 L of buffer C (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, and 5 mM 2-mercaptoethanol) containing 50 mM NaCl (buffer D) at 4° C. The dialyzed proteins (200 mg protein) were applied to a DEAE-Sepharose CL-6B column (26.4 400 mm) pre-equilibrated with buffer D at a flow rate of 1.0 ml/min. The fraction size was 10 ml/tube. Aliquots (50 ll) of the fractions were used to assay for calpain activity as described [5]. Unbound proteins, containing mitochondrial m-calpain, were eluted with 700 ml of buffer D. Mitochondrial l-calpain was eluted with a linear gradient of 50– 300 mM NaCl in buffer C in a total volume of 1.1 L. Each fraction was used for the calpain assay in the presence of the OM proteins (Fig. 2B). Fifty micrograms of OM proteins were added to 50 ll of each fraction and incubated at 4 °C for 2 h. In contrast, 1% octylglucoside-solubilized OM proteins were added to each fraction and incubated. Then the calpain activity of each sample was measured as described [5].
Solubilization and column chromatography of mitochondrial OMbound calpains
Partial purification of calpain small subunit in mitochondrial IMS
Mitochondrial OM was prepared from 10 Sprague–Dawley (SD) rats (8 weeks-old) as described above. To prevent the contamination of cytosolic proteins, the mitochondrial surface was treated with proteinase K as described in detail [7]. DEAE-Sepharose CL-6B (Amersham Pharmacia Biotech) column chromatography was used to separate the calpain isoforms in the OM (Fig. 2A). The prepared OMs were suspended in solubilizing buffer (20 mM Tris– HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 5 mM 2-mercaptoethanol, 1% octyl-glucoside, and 50 mM NaCl). The samples solubilized for 30 min at 4 °C and were then centrifuged at 20,000g for 30 min. The supernatants (360 mg protein) were applied to a DEAESepharose CL-6B column (26.4 400 mm) pre-equilibrated with buffer A (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 5 mM 2-mercaptoethanol, 0.1% octyl-glucoside) containing 50 mM NaCl (buffer B) at a flow rate of 1.0 ml/min. All chromatographic procedures were performed at 4 °C. The amount of each fraction size was 10 ml/tube. Aliquots (50 ll) of the separated fractions were used to assay for calpain activity as described [5].
Sepharose 6B column chromatography (Amersham Pharmacia Biotech) was used to partially purify the calpain small subunit from the mitochondrial IMS (Fig. 4A). Rat liver mitochondrial IMS were prepared from 30 SD rats (8 weeks-old) as described above. The prepared IMS fraction was dialyzed overnight against 2 L of buffer C (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, and 5 mM 2-mercaptoethanol) containing 140 mM NaCl (buffer E) at 4 °C. The dialyzed proteins (20 mg protein) were applied to a Sepharose column (9 600 mm) pre-equilibrated with buffer E at a flow rate of 0.5 ml/min. The proteins were eluted with buffer E depending on their molecular weight. The molecular weight markers were normal rabbit IgG (140 kDa), BSA (66 kDa), and chymotrypsinogen A (24 kDa). The fraction size was 500 ll/tube. Each fraction was concentrated by Amicon PM-10 membrane (Millipore Co., Bedford, MA). The concentrates were used for Western blot analysis with anti-l- and anti-m-calpain large subunits, anticalpain small subunit, anti-ERp57, and anti-Grp75 antibodies (Fig. 4B).
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Western blotting Western blotting was performed as in our earlier studies [5–7]. After electrophoretic transfer of the proteins to a nitrocellulose membrane, the membranes were treated with the primary antibodies followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rabbit anti-goat IgG secondary antibodies (DAKO, Cambridge, UK). The immunoreactive signals were developed with an ECL Western blotting detection kit (Amersham Biosciences) and quantified with a Luminescent Image Analyzer, LAS-3000 (Fujifilm Co., Tokyo, Japan). Assay of ATP concentrations in SD rats liver mitochondrial and cytosolic fractions To determine the concentration of ATP in SD rat liver mitochondrial and cytosolic fractions, we used an ATP measurement kit (Bio Assay Systems, CA). This kit provides a rapid method to measure intracellular cytosolic or organelle ATP. In the presence of luciferase, ATP immediately reacts with the Substrate D-luciferin to produce light. The light intensity is a direct measure of ATP concentration. We measured ATP concentration of SD rat liver, kidney, heart, and brain cytosolic and mitochondrial fractions following a manual method of the kit. The ATP concentration of rat liver mitochondrial and cytosolic fractions were 1.3 and 1.5 mM, respectively. The concentrations of both kidney mitochondrial and cytosolic fractions were 1.1 mM. In heart and brain, ATP concentrations were relatively high (2–4 mM). Effects of calpain small subunit, Grp75, and ATP on calpain activity in mitochondrial OM Mitochondrial OM fractions were prepared from SD rat (8 weeks-old) liver and suspended in buffer E. The samples (100 lg protein/tube) were incubated at 4 °C for 30 min with or without Sepharose 6B-purified calpain small subunit (10 lg protein), Grp75 protein (1 lg protein), 2 mM final concentrations of ATP, TTP, GTP, and CTP. Then the calpain activity of each sample was assayed (Fig. 5A). Effects of Ca2+, ATP, calpain small subunit, and Grp75 on releases of mitochondrial calpains from OM Mitochondrial OM fractions were prepared from SD rat (8 weeks-old) liver, and the prepared OM was suspended in buffer E. The samples (200 lg protein/tube) were incubated at 30 °C for 3 min with or without Sepharose 6B-purified calpain small subunit (10 lg protein), Grp75 protein (1 lg protein), 2 mM final concentrations of ATP, TTP, GTP, CTP, 50 lM Ca2+ or 500 lM Ca2+. Each sample was centrifuged at 100,000g for 1 h and the supernatants were concentrated with Amicon PM-10 membrane. The concentrates were used for Western blot analyses with anti-l-calpain and m-calpain large subunits (Fig. 5B). Ca2+ requirement of m-calpain release from OM SD rat liver mitochondrial OM was suspended in buffer E. The samples (200 lg protein/tube) were incubated at 30 °C for 3 min with Sepharose 6B-purified calpain small subunit (10 lg protein), Grp75 protein (1 lg protein), 2 mM ATP and 50 lM PD150606 in the presence of different concentrations of CaCl2 (0–1000 lM). Each sample was centrifuged at 100,000g for 1 h, and the supernatants were concentrated with Amicon PM-10 membrane. The concentrates were used for Western blot analyses with antim-calpain large subunits (Fig. 5C). Immunoreactive bands were
quantified using a LAS-3000, luminescent image analyzer (Fujifilm Co., Tokyo, Japan). Ca2+ dependency of mitochondrial m-calpain activity The Ca2+ dependency of mitochondrial m-calpain was determined using DEAE-Sepharose-purified IMS m-calpain as described [7] (Fig. 5C). The samples (50 lg protein) were added to 450 ll of assay solution (100 mM Tris–HCl, pH 7.5, 10 mM 2-mercaptoethanol, 100 mM KCl, 1 mM EGTA, 20 lM Succ-Leu-Tyr-AMC) in the presence of different concentrations of CaCl2 (0–1000 lM) and incubated at 25 °C. The continuous methods of calpain assay were performed as described in detail [5]. Immunoprecipitation analyses of Grp75-m-calpain released from OM interaction We also performed immunoprecipitation analyses to determine whether the mitochondrial m-calpain released from OM form complexes with calpain small subunit and Grp75 (Fig. 5D). The prepared OMs (200 lg protein/tube) were incubated at 30 °C for 3 min with Sepharose 6B-purified calpain small subunit (10 lg protein), Grp75 protein (1 lg protein), 2 mM final concentrations of ATP, and 50 mM Ca2+. The samples were centrifuged at 100,000g for 1 h. Polyclonal antibody to Grp75 (1 lg) was incubated with the supernatants containing mitochondrial m-calpain released from the OM in buffer E. Normal rabbit IgG was used as a control for the anti-Grp75 antibody. After standing for 18 h at 4 °C, excess protein G-Sepharose 4 Fast Flow was added (12 ll/ tube), and allowed to stand for 2 h at 4 °C. The preparations were centrifuged at 12,000 rpm for 5 min at 4 °C. The pellets were washed six times with 50 mM Tris–HCl, pH 7.4, containing 140 mM NaCl, and 0.1% Emulphogen BC 720. The co-immunoprecipitated proteins were analyzed by Western blot analyses with anti-m-calpain large subunit and anti-calpain small subunit. Results Activation of mitochondrial OM-bound calpains by different detergents The calpain specific activity of non-solubilized OM was 60 FI/min/mg, and it increased fivefold after solubilization with digitonin, twofold with CHAPS, and threefold with Tween and octylglucoside (Fig. 1). The increased calpain activities were inhibited by 95% by PD150606, a calpain specific inhibitor. Addition of IMS proteins to octyl-glucoside-solubilized OM proteins increased the calpain specific activities by sixfold compared to non-treated IMS fraction (data not shown). This increase in the calpain activity was inhibited by PD150606 by 90%. These results indicated that the IMS proteins activated the OM-bound calpains or the OM proteins activated the IMS calpains. We also examined whether OM proteins activated IM calpain or IM proteins activated OM calpains by mixing each fraction. However, the OM and IM calpain activities were not changed (data not shown). These findings suggested that the activator of OM calpains is not localized in the IM or that of IM calpains is not present in the OM. Effects of OM proteins on mitochondrial l- and m-calpains in mitochondrial IMS We next examined whether the mitochondrial OM proteins were activated the mitochondrial l- and m-calpains of the IMS (Fig. 2). Mitochondrial l- and m-calpains were separated by DEAE-Sepharose CL-6B column chromatography as described [7].
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Fig. 1. Effects of different detergents and IMS proteins on the activation of mitochondrial calpains in OM. Mitochondrial OM proteins were solubilized with a 1% final concentration of sodium deoxycholate, digitonin, CHAPS, Nonidet p-40 (NP40), n-dodecyl-b-D-maltoside (DDM), Triton X-100 (TX-100), Tween and octylglucoside. Each solubilized protein was isolated by centrifugation as described in the Materials and methods section. The samples solubilized with each detergent were assayed for calpain activity. The calpain specific activity of non-solubilized OM is 60 FI/min/mg. The activities increased by about fivefold after solubilization with digitonin, twofold with CHAPS, and threefold with Tween and octyl-glucoside. The increased activities caused by the solubilization with several detergents were inhibited by 95% by the calpain specific inhibitor, PD150606. Data are expressed as means ± SD n = 3.
We obtained three peaks of calpain activity; an unbound protein fractions (Peak 1), a peak at 120 mM NaCl (Peak 2), and a peak at 150 mM NaCl (Peak 3; Fig 2A). We found that Peak 1 contained mitochondrial m-calpain, Peak 2 contained mitochondrial l-calpain, and Peak 3 contained ERp57-associated mitochondrial l-calpain [7]. Unsolubilized OM proteins were added to each fraction but no further activation of mitochondrial l- and m-calpains was found (Fig. 2B). These results demonstrated that mitochondrial OM proteins do not affect the specific activities of mitochondrial l- and m-calpains in the IMS. In addition, these data suggested that the l- and m-calpains located in the IMS do not affect the activity of OM-bound calpain. In contrast, the addition of octylglucoside-solubilized OM proteins increased the calpain activities and a new peak appeared (Fig. 2B). We detected the calpain small subunit in the new peak by Western blot analysis (data not shown). We suggest that the calpain small subunit in IMS activate octyl-glucoside-solubilized OM-bound calpains.
Identification of calpain isoforms in mitochondrial OM We separated the mitochondrial OM-bound calpains by DEAESepharose CL-6B column chromatography. We solubilized the OM proteins with 1% octyl-glucoside and applied it to the column. The unbound proteins were eluted with buffer B, and the adsorbed proteins were eluted with a linear gradient of 50–300 mM NaCl in buffer A (Fig. 3A). We detected four peaks of calpain activity; an unbound protein fraction (Peak 1), a peak for 100 mM NaCl (Peak 2), a peak for 125 mM NaCl (Peak 3), and a peak for 200 mM NaCl (Peak 4). Each peak was collected and concentrated. Western blot analyses showed that the autolyzed forms of l-calpain large subunit and calpain small subunit were contained in Peak 1, and the intact forms of l-calpain and m-calpain large subunits in Peak 4 (Fig. 3B). Interestingly, the calpain small subunit and Grp75 were not detected in the fraction containing l-calpain and m-calpain large subunits. We detected mitochondrial calpain-associated chaperon ERp57 in Peaks 1, 3, and 4. Grp75, a protein associated with mitochondrial m-calpain, was present in Peak 3. We obtained calpain-like protease activities in Peaks 2 and 3 as shown in Fig. 3A, although no calpain large and small subunits
Fig. 2. Effects of OM proteins on mitochondrial l- and m-calpains in IMS. (A) Mitochondrial l- and m-calpains were separated by DEAE-Sepharose CL-6B column chromatography using the IMS fraction as described in Materials and methods section. Three peaks of calpain activity can be seen: an unbound protein fractions (Peak 1), a peak at 120 mM NaCl, and a peak at 150 mM NaCl (Peak 3). Peak 1 contains the Grp75-associated mitochondrial m-calpain, Peak 2 contains mitochondrial l-calpain, and Peak 3 contains ERp57-associated mitochondrial lcalpain. (B) Unsolubilized OM proteins and 1% octyl-glucoside-solubilized OM proteins were added to each fraction and incubated at 4 °C for 2 h. Details are described in the Materials and methods section. No significant increase in the calpain activity is observed in unsolubilized OM proteins-added fractions. Addition of octyl-glucoside-solubilized OM proteins increases the calpain activities and a new peak appeared. Results are representative of three independent experiments (A and B).
were detected as shown in Fig. 3B. So we checked the activities of Peaks 2 and 3 with calpain specific inhibitor, PD150606, and we found that PD150606 did not inhibit the activities (data not shown). In addition, we found that the activities of Peaks 2 and 3 were obtained in the absence of Ca2+. The calpain substrate we used in the assay, Suc-Leu-Tyr-AMC, was also cleaved by cathepsins to some degree [15]. Therefore, these results suggested that Peaks 2 and 3 contained cathepsin-like proteases. Immunoprecipitation analysis showed that the 55–65 kDa autolyzed l-calpain large subunit did not associate with the 30 kDa calpain small subunit in Peak 1, and the 80 kDa l-calpain large subunit bound to ERp57 in Peak 4 (Fig. 3C). We also examined whether the mitochondrial IMS proteins activated the OM-bound calpains. The calpains of peak 4 containing l- and m-calpain large subunits were significantly activated by IMS proteins (Fig. 3D).
Presence of calpain small subunit in mitochondrial IMS Earlier, we found an unknown calpain-associated small subunit or free small subunit in the IMS. We expected that the free calpain small subunit was involved in the activation of the OM-bound calpains. We confirmed that the free calpain small subunit was
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Fig. 3. Identification of calpain isoforms in mitochondrial OM and their activations with IMS proteins. (A) The OM proteins were separated with DEAE-Sepharose CL-6B column chromatography. Details are described in the Materials and methods section. The octyl-glucoside-solubilized OM proteins were applied to the column. Four peaks of calpain-like activity are present; an unbound protein fractions (Peak 1), at 100 mM NaCl (Peak 2), at 125 mM NaCl (Peak 3), and at 200 mM NaCl (Peak 4). (B) Western blot analyses of each peak of calpain-like activity (30 lg protein/lane). From top to bottom panel, antibodies used were anti-l-calpain large subunit, m-calpain large subunit, calpain small subunit, ERp57, and Grp75 antibodies. The IMS fraction (20 lg protein) was used as controls of ERp57 and Grp75 (far right lane). Peak 1 contains autolyzed lcalpain large subunit, calpain small subunit and ERp57. Peak 3 contains ERp57 and Grp75. Peak 4 contains the intact l-calpain, m-calpain large subunit and ERp57 but not calpain small subunit. (C) Immunoprecipitation analyses of mitochondrial l-calpain with anti-calpain small subunit and anti-ERp57 antibodies. Details are described in the Materials and methods section. The 55–65 kDa autolyzed l-calpain large subunit did not associate with the 30 kDa calpain small subunit in Peak 1, and the 80 kDa l-calpain large subunit bound to the ERp57 in Peak 4. Results are representative of three independent experiments. (D) Effects of IMS proteins on each peak. Details are described in the Materials and methods section. In the presence of IMS proteins, only Peak 4, which contains l-calpain and m-calpain large subunits, is activated twofold to non-treated sample. Results are representative of three independent experiments.
present in the IMS by Sepharose 6B gel filtration column chromatography (Fig. 4A). Each fraction was concentrated by Amicon PM10 membrane and used for Western blot analyses of the calpain small subunit. The calpain small subunit was contained in the >140 kDa, 110 kDa, and 24–30 kDa protein fractions (Fig. 4B). The >140 kDa protein fraction contained ERp57-associated lcalpain and Grp75-associated mitochondrial m-calpains, while the 110 kDa protein fraction contained l-calpain. ERp57 and Grp75 were detected in different fractions. We detected the small subunit in the fractions that did not contain calpain activities, and the large subunits of l- and m-calpains in the 24–30 kDa protein fractions. These results indicated that part of the calpain small subunits is free in the IMS. To confirm the effects of the free calpain small subunits on the activity of the OM-bound calpains, we added the Sepharose 6B-purified calpain small subunit to octyl-glucoside-solubilized OM proteins and incubated the mixture at 4 °C for 2 h. The calpain small subunit significantly activated the OM-bound calpains (Fig. 4C). The calpain small subunit that had been exposed to anti-calpain small subunit did not activate the OM-bound calpains, and the boiled small subunit also did not activate the OM-bound calpains. These results showed that the free calpain small subunit modulated the activity of the OM-bound calpains. Effects of calpain small subunit, Grp75, and ATP on calpain activity in mitochondrial OM The results shown in Figs. 1–4 suggested that the free calpain small subunit can activate solubilized OM-bound calpains. However, the results obtained on solubilized OM do not necessarily
reflect its activity in the mitochondria. To examine this, we used unsolubilized OM and observed the effects of calpain small subunit, Grp75, and ATP on the calpain activities in the unsolubilized OM. We added free calpain small subunit, Grp75, ATP, GTP, CTP, and TTP to the unsolubilized OM under different conditions. The samples were incubated at 4 °C for 30 min, and the calpain activity was assayed. The calpain specific activity of non-solubilized OM was 60 FI/min/mg (Fig. 5A). The activity was twice as high as the non-treated OM in the presence of calpain small subunit. Grp75 did not affect the activity, but the addition of the small subunit and Grp75 increased the activity by about threefold compared with the non-treated condition. In addition, we detected an increase in the calpain activity by fivefold over non-treated OM in the presence of small subunit, Grp75, and ATP. However, it appeared that the calpain activity in the OM remained unaffected by the presence of TTP, GTP, and CTP. Even if the Grp75 protein was not added to the OM, the activity increased threefold over the non-treated one in the presence of the small subunit and ATP. These data indicated that the OM-bound calpain large subunits were activated by Grp75 and ATP as well as the calpain small subunit. Effects of Ca2+, ATP, calpain small subunit, and Grp75, and ATP on releases of mitochondrial calpains from OM The mitochondrial unsolubilized OM-bound calpains were activated by Grp75, calpain small subunit, and ATP (Fig. 5A). It was not certain whether mitochondrial calpains are activated on the OM or after being released from the OM. Therefore, we examined whether the OM-bound m-calpain was released from the OM and then
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activated (Fig. 5B). We added the 50 lM Ca2+, 500 lM Ca2+, ATP, the free calpain small subunit, and Grp75 to the unsolubilized OM in different combinations. The samples were then incubated at 30 °C for 3 min and centrifuged at 100,000g for 1 h to obtain the proteins released from the OM. The supernatants containing the proteins released from the OM were analyzed by Western blotting for l-calpain and m-calpain large subunits. Mitochondrial m-calpain large subunit was released from the OM in the presence of 50 lM Ca2+, ATP, the calpain small subunit, and Grp75. High concentration of Ca2+ (500 lM Ca2+) induced the release of the autolytic products of m-calpain large subunit as well as the intact forms. Without Ca2+, mitochondrial m-calpain large subunit was minimally released. The release of m-calpain large subunit was weakly inhibited without ATP. The release was blocked by the absence of the calpain small subunit and Grp75. Addition of 50 lM Ca2+ alone induced a slight release of m-calpain large subunit. ATP alone, small subunit alone, or Grp75 alone did not induce the release. On the other hand, the l-calpain large subunits were not released from the OM under all conditions. Ca2+ requirement of m-calpain releases from OM We examined the effects of Ca2+ concentrations on the releases of the m-calpain large subunit from the OM by Western blotting (Fig. 5C). Immunoreactive bands of released m-calpain large subunit were quantified using a luminescent image analyzer. The m-calpain large subunits began to be released from the OM at 25 lM Ca2+, and 40% of these were released at 50 lM Ca2+. However, almost all IMS m-calpain was not activated at 25–100 lM Ca2+. These results suggested that the release occurred before m-calpain was activated. Immunoprecipitation analyses of Grp75-m-calpain released from OM interaction Immunoprecipitation analyses showed that the m-calpain large subunit released from the OM formed complexes with the calpain small subunit and Grp75 (Fig. 5D). Discussion
Fig. 4. Presence of a free calpain small subunit in IMS. Details are described in Materials and methods section. (A) Sepharose 6B column chromatography of mitochondrial IMS proteins. Molecular weight markers used were normal rabbit IgG (140 kDa), BSA (66 kDa) and chymotrypsinogen A (24 kDa). (B) Western blot analysis of l- and m-calpain large subunits, calpain small subunit, ERp57 and Grp75 using Sepharose 6B-separated IMS fractions (20 lg protein/lane). We detected the calpain small subunit in the >140, 110, and 24–30 kDa protein fractions. The circled fractions in panel A contain the calpain small subunit. The >140 kDa protein fraction contains ERp57-associated l-calpain and Grp75-associated mitochondrial m-calpains, and the 110 kDa protein fraction contains l-calpain. The calpain small subunit was also detected in the 24–30 kDa protein fractions. These results indicate that a part of calpain small subunits are free in the IMS. (C) Effects of the free calpain small subunit on the activity of mitochondrial OM-bound calpains. The Sepharose 6B-purified calpain small subunit was incubated with octylglucoside-solubilized OM proteins at 4 °C for 2 h. The calpain small subunit significantly activated the OM-bound calpains (250% compared to the non-treated OM proteins). Absorption of calpain small subunit with anti-calpain small subunit antibody does not activate the OM-bound calpains. The partial purified calpain small subunit was boiled at 80 °C for 3 min and incubated with OM proteins. An increase of calpain activity by the boiled small subunit was not detected. Data are expressed as means ± SD, n = 3, ⁄P < 0.01 vs the calpain specific activity of nontreated condition.
Our results showed that IMS-localized free calpain small subunit and Grp75 play critical roles for activating OM-bound m-calpain. They also showed that pre-activated mitochondrial m-calpain large subunit is released from the OM together with the calpain small subunit and Grp75 by exposure to low concentrations of Ca 2+. After their releases from the OM, mitochondrial m-calpain appeared to be further activated by the increased mitochondrial Ca2+ level. We previously suggested that OM-bound calpains existed as the pre-active forms under physiological condition [5]. This is probably because the OM-bound mitochondrial l- and m-calpains are not bound to the calpain small subunit (Fig. 3). The mitochondrial m-calpain domain IV, which is associated with the calpain small subunit, is probably embedded in the OM. This would then suggest that the OM-bound mitochondrial m-calpain large subunit does not bind to the small subunit and has weak activity. The OM-bound calpains are activated by several detergents such as digitonin, Tween, and octyl-glucoside (Fig. 1). This indicates that the solubilization of OM-bound calpains allows the calpains to change their conformation from the pre-active to the active form. In the case of cytosolic m-calpain, the small subunit plays an essential role in the conformational change to the active form [16,17]. The restriction of the conformational changes of the
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Fig. 5. Effects of Ca2+, calpain small subunit, Grp75 and ATP on calpain activity of mitochondrial OM-bound calpains and their releases from OM. Details are described in Materials and methods section. (A) Effects of calpain small subunit, Grp75, ATP, TTP, GTP, and CTP on the activity of mitochondrial OM-bound calpains. The calpain specific activity of non-solubilized OM was 60 FI/min/mg (column 1). The calpain small subunit activated the OM-bound calpains twice as high as the non-treated OM (column 2). The activity is not changed by the presence of Grp75 (column 3). The addition of calpain small subunit and Grp75 induces an increase in the activity by about threefold of the non-treated one (column 4). Further addition of ATP activated the OM-bound calpains over sixfold as high as non-treated one (column 5). TTP, GTP, and CTP had no significant effects for the OM-bound calpain activities (columns 6–8). The OM-bound calpains were not activated in the presence of only ATP (column 9). The addition of calpain small subunit and ATP induced the activation about threefold of the non-treated one (column 10). Without calpain small subunit, the OM-bound calpains are not activated (column 11). All data are mean ± SD, n = 3, ⁄P < 0.01, ⁄P < 0.001 vs the calpain specific activity of non-treated OM. (B) Effects of Ca2+, ATP, calpain small subunit, and Grp75 on releases of mitochondrial calpains from OM. Mitochondrial m-calpain large subunit was released from the OM in the presence of 50 lM Ca2+, ATP, calpain small subunit, and Grp75 (lane 1). High concentration of Ca2+ (500 lM) induced the releases of the intact and autolyzed mitochondrial m-calpain large subunit in the presence of ATP, calpain small subunit, and Grp75 (lane 2). Mitochondrial m-calpain large subunit was not released from the OM without Ca2+ (lane 3). Without ATP, their releases weakened slightly (lane 4). The releases were strongly blocked without calpain small subunit (lane 5) and Grp75 (lane 6). Addition of 50 lM Ca2+ alone induced a slight release of m-calpain large subunit (lane 7). ATP alone, small subunit alone, or Grp75 alone did not induce the release (lane 8–10). In contrast, mitochondrial l-calpain large subunit was not released from the OM under all conditions (lower panel). Mitochondrial OM was used for the positive controls of l-calpain and m-calpain large subunits (lane 11, 40 lg protein). Results are representative of three independent experiments. (C) Ca2+ requirements for the release of m-calpain from the OM and Ca2+ dependency of mitochondrial m-calpain activities. We examined the correlation between Ca2+ requirement for m-calpain releases from the OM and Ca2+ dependency of mitochondrial m-calpain activities. The m-calpain large subunits begin to be released from the OM at 25 lM Ca2+, which increases to 40% at 50 lM Ca2+. However, almost all IMS m-calpain was not activated at 25–100 lM Ca2+. Data are the means ± SD, n = 3. (D) Immunoprecipitation of mitochondrial m-calpain released from OM with anti-Grp75 antibody. Both mitochondrial m-calpain large (open arrowhead) and small subunits (solid arrowhead) are co-precipitated with Grp75. The results show that mitochondrial m-calpain large subunits released from the OM formed the complexes with calpain small subunits and Grp75.
OM-bound m-calpain large subunit is manifested by the weak activity of OM-bound calpains. Another possibility is that permeabilization of OM proteins dissociated the OM-bound calpains from their binding partners such as the calpastatin-like inhibitory proteins. At present, we are trying to identify the binding partners by column chromatography, immunoprecipitation with anti-l- and m-calpain large subunits antibodies, and LC–MS/MS analyses. We found that Ca2+ and the free calpain small subunit release the mitochondrial m-calpain large subunit from the OM and activate it (Figs. 4 and 5). The release occurred at a low concentration of Ca2+, which does not activate the mitochondrial m-calpain (Fig. 5B and C), and the released mitochondrial m-calpain combines with the small subunit (Fig. 5D). In contrast, a high concentration of Ca2+ (500 lM Ca2+) causes the autolysis of mitochondrial m-calpain, indicating that it is activated (Fig. 5B). We suggest that the low concentrations of Ca2+ induced conformational changes of the OM-bound m-calpain large subunit but stops short of its activation. It is likely that the conformational change exposes its domain IV to the IMS and leads to the binding of the free calpain small subunit. This heterodimer formation may induce its release from the OM into the IMS. Additional Ca2+ influx would activate
the mitochondrial m-calpain released from the OM. Another important change induced by Ca2+ was the conformational change of the free calpain small subunit in the IMS. The calpain small subunit has Ca2+ binding sites in its domain VI [1]. The conformational change of the free calpain small subunit may increase its accessibility of the subunits to OM-bound m-calpain large subunit to form the heterodimer. Not only Ca2+ and the free calpain small subunit but also Grp75 play significant roles in the release and activation of OM-bound mitochondrial m-calpain large subunit (Fig. 5). Grp75 plays a role in the import into the mitochondria and in the proper folding of nuclear encoded proteins in an ATP-dependent manner [18,19]. We previously found that m-calpain bound to Grp75 in mitochondrial IMS [7]. In contrast, the present study showed that the mcalpain large subunit does not bind to both small subunit and Grp75 in the OM. We demonstrated that the molecular forms of calpains are mostly different between IMS and OM. The OMlocation of l- and m-calpains under imperfect conditions such as l-calpain large subunit/ERp57 and m-calpain large subunit alone indicates that the OM is mitochondrial calpains settlement until they are supplied to the IMS and are activated. The present study
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indicated that Grp75 promotes the release and activation of OM-bound mitochondrial m-calpain. We suggested that a portion of OM-bound m-calpain large subunit is unfolded, and Grp75 binds to the subunits and makes it properly folded. It is unclear whether Grp75 binds to the OM-bound m-calpain large subunit and then the calpain small subunit associates the calpain. However, this proper folding seems to stimulate the release of mitochondrial m-calpain from the OM. Mitochondrial m-calpain was not released from the OM without Ca2+ in the presence of the calpain small subunit, Grp75, and ATP (Fig. 5B). These findings suggest that Ca2+ causes conformational changes of the OM-bound m-calpain large subunits and then Grp75 chaperones bind to the subunits and pull them into the IMS together with the small subunits. Our results demonstrated that the activation of OM-bound mitochondrial m-calpain occurs in several steps: (1) an apoptotic stimulus leads to the influx of Ca2+ into the cytosol; (2) the prolonged increase of the intracellular Ca2+ level leads to mitochondrial uptake of Ca2+; (3) conformational changes of mitochondrial m-calpain large subunit in the OM occur in response to the Ca2+; (4) the conformational changes lead to the binding of calpain small subunit and Grp75 to OM-bound mitochondrial m-calpain large subunit; (5) this complex formation causes the release of the mitochondrial m-calpain from the OM into the IMS; and (6) a further increase of mitochondrial Ca2+ level activates the mitochondrial m-calpain. The fundamental finding of this study was that OM-bound mitochondrial m-calpains are released to the IMS with the calpain small subunit and Grp75 by influx of Ca2+. We previously suggested that mitochondrial m-calpain in the IMS promotes an apoptotic cell death by releasing tAIF into the cytosol through VDAC–Bax complexes [7]. The increase of mitochondrial m-calpain released from OM must facilitate apoptosis by releasing of tAIF into the cytosol. The present study provides a new insight that the specific inhibition of mitochondrial m-calpain has a therapeutic
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potential for the many AIF-mediated disorders such as retinitis pigmentosa and cerebral ischemia. Acknowledgments The authors thank Prof. Duco Hamasaki for careful editing and Prof. Hideaki Kikuchi for helpful advice. This work was done in part at Gene Research Center, Hirosaki University. References [1] D.E. Goll, V.F. Thompson, H. Li, W. Wei, J. Cong, Physiol. Rev. 83 (2003) 731–801. [2] K. Suzuki, S. Hata, Y. Kawabata, H. Sorimachi, Diabetes 53 (Suppl 1) (2004) S12–18. [3] P. Kar, K. Samanta, S. Shaikh, A. Chowdhury, T. Chakraborti, S. Chakraborti, Arch. Biochem. Biophys. 495 (2010) 1–7. [4] S. Endo, S. Ishiguro, M. Tamai, Biochim. Biophys. Acta 1450 (1999) 385–396. [5] T. Ozaki, H. Tomita, M. Tamai, S. Ishiguro, J. Biochem. 142 (2007) 365–376. [6] T. Ozaki, T. Yamashita, S. Ishiguro, Biochim. Biophys. Acta 1783 (2008) 1955– 1963. [7] T. Ozaki, T. Yamashita, S. Ishiguro, Biochim. Biophys. Acta 1793 (2009) 1848– 1859. [8] S. Mizukoshi, M. Nakazawa, K. Sato, T. Ozaki, T. Metoki, S.I. Ishiguro, Exp. Eye Res. 91 (2010) 353–361. [9] G. Cao, J. Xing, X. Xiao, A.K. Liou, Y. Gao, X.M. Yin, R.S. Clark, S.H. Graham, J. Chen, J. Neurosci. 27 (2007) 9278–9293. [10] P.S. Vosler, D. Sun, S. Wang, Y. Gao, D.B. Kintner, A.P. Signore, G. Cao, J. Chen, Exp. Neurol. 218 (2009) 213–220. [11] E. Norberg, V. Gogvadze, H. Vakifahmetoglu, S. Orrenius, B. Zhivotovsky, Free Radic. Biol. Med. 48 (2010) 791–797. [12] T. Tamura, T. Yamashita, H. Segawa, H. Taira, FEBS Lett. 513 (2002) 153–158. [13] D.F. Parsons, G.R. Williams, B. Chance, Ann. N Y Acad. Sci. 137 (1966) 643–666. [14] M.M. Bradford, Anal. Biochem. 72 (1976) 248–254. [15] T. Sasaki, T. Kikuchi, N. Yumoto, N. Yoshimura, T. Murachi, J. Biol. Chem. 259 (1984) 12489–12494. [16] J.S. Elce, C. Hegadorn, J.S. Arthur, J. Biol. Chem. 272 (1997) 11268–11275. [17] J.S. Elce, P.L. Davies, C. Hegadorn, D.H. Maurice, J.S. Arthur, Biochem. J. 326 (Pt 1) (1997) 31–38. [18] L.A. Mizzen, C. Chang, J.I. Garrels, W.J. Welch, J. Biol. Chem. 264 (1989) 20664– 20675. [19] L.A. Mizzen, A.N. Kabiling, W.J. Welch, Cell Regul. 2 (1991) 165–179.
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Archives of Biochemistry and Biophysics journal homepage: www.elsevier.com/locate/yabbi
Ca2+-induced release of mitochondrial m-calpain from outer membrane with binding of calpain small subunit and Grp75 Taku Ozaki a, Tetsuro Yamashita b, Sei-ichi Ishiguro a,⇑ a b
Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japan Department of Food Science and Biochemistry, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan
a r t i c l e
i n f o
Article history: Received 21 October 2010 and in revised form 30 November 2010 Available online 9 December 2010 Keywords: Calpain Calpain small subunit Grp75 Activation Mitochondrial outer membrane Apoptosis
a b s t r a c t Although mitochondrial l- and m-calpains play significant roles in apoptotic cell death, their activating mechanisms have not been determined. The purpose of this study was to determine the core factors that are involved in activating mitochondrial outer membrane (OM)-bound calpains. To accomplish this, we solubilized OM-bound calpains and separated them by DEAE-Sepharose column chromatography, and identified them by immunoblots. We also determined the core factors that activated the OM-bound calpains and release them from the OM by calpain assays, immunoprecipitations, and immunoblots. The OM-bound m-calpain large subunit was not associated with the small subunit or with Grp75 chaperone. Free calpain small subunit was located in the IMS and caused the release of the OM-bound m-calpain large subunit from the OM together with Grp75, ATP, and Ca2+. Our results showed that the activating mechanism of mitochondrial OM-bound m-calpain and the release of mitochondrial m-calpain from the OM have important implications in facilitating apoptotic cell death. Published by Elsevier Inc.
Introduction Calpains are Ca2+-activated neutral cysteine proteases that modulate the functions of many specific substrates by limited proteolysis [1]. In humans, 14 calpain isoforms have been identified as the catalytic large subunit and two isoforms for regulatory small subunits [1,2]. Among them, l-calpain and m-calpain large subunits, calpain small subunit, and calpain 10 exist in mitochondria [3]. The mitochondrial calpains play significant roles in pathophysiological conditions including apoptotic and necrotic cell deaths [3]. Endo et al. showed that mitochondrial calpains inactivate mitochondrial aspartate aminotransferase in the rat retina under ischemic and hypoxic conditions [4]. We found that mitochondrial l-calpain modulates apoptotic cell death by limiting the cleavage of the apoptosis-inducing factor (AIF)1 [5,6]. We also demonstrated that mitochondrial m-calpain plays a significant role in the
⇑ Corresponding author. Address: Division of Cell Technology, Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki 036-8561, Japan. Fax: +81 172 39 3780. E-mail address: [email protected] (S.-i. Ishiguro). 1 Abbreviations used: AIF, apoptosis-inducing factor; tAIF, truncated AIF; VDAC, voltage-dependent anion channel; Grp75, glucose-regulated protein 75; GAPDH, glyceraldehyde phosphate dehydrogenase; OM, outer membrane; IMS, intermembrane space; IM, inner membrane; ATP, adenosine 50 -triphosphate; TTP, thymidine 50 triphosphate; GTP, guanosine 50 -triphosphate; CTP, cytidine 50 -triphosphate. 0003-9861/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.abb.2010.12.003
release of truncated AIF from mitochondria through VDAC–Bax complex [7]. Very recently, we proved that the mitochondrial calpains induce photoreceptor apoptosis in the Royal College of Surgeon’s (RCS) rats, which are widely employed as an animal model of human retinitis pigmentosa [8]. Although mitochondrial calpains are involved in a variety of pathological conditions [8–11], how they are activated has not been determined. Earlier, we found that the mitochondrial mcalpain large subunit was located mainly in the OM [7], but the calpain activity in the OM was very weak [5]. These observations suggested that mitochondrial m-calpain in the OM is regulated by not only Ca2+ but also other factors such as calpain activators, inhibitors, and molecular chaperones. The purpose of this study was to determine the core factors that are involved in activating OM-bound calpains. We have shown that mitochondrial l- and m-calpains are associated with the molecular chaperones such as ERp57 and Grp75, respectively, in the mitochondrial intermembrane space (IMS) [6,7]. In addition, we detected three types of calpain small subunits in the IMS using non-denaturing gels: the first was a mitochondrial l-calpain large subunit-associated calpain small subunit, the second was a mitochondrial m-calpain-associated calpain small subunit, and the third was an unknown calpain large subunit-associated or free calpain small subunit [6]. To identify the activating factors of OM-bound calpains, we determined what kinds of calpain isoforms were present in the OM and investigated the effects of Ca2+, ERp57, Grp75, and calpain small subunit.
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Materials and methods Materials The detergents used to solubilize the mitochondrial outer membrane (OM) were: sodium deoxycholate (Fluka, Milwaukee, WI), Nonidet P-40 and Octyl-glucoside (Sigma Aldrich Corp., St. Louis, MO), digitonin, CHAPS, n-dodecyl-b-D-maltoside, Triton X-100, and Tween 20 (Wako). Grp75 protein was purchased from Abcam (Cambridge, MA). Adenosine 50 -triphosphate (ATP), thymidine 50 triphosphate (TTP), guanosine 50 -triphosphate (GTP) and cytidine 50 -triphosphate (CTP) were purchased from Sigma. The antibodies purchased were: rabbit polyclonal antibodies against l-calpain large subunit domain IV (Sigma); anti-m-calpain large subunit domain III (Abcam); anti-calpain small subunit domain V (Abcam); anti-Grp75 (Santa Cruz Biotechnology Inc., CA); anti-adenylate kinase 2 (Santa Cruz); anti-GAPDH (Santa Cruz); anti-VDAC (Calbiochem); anti-pyruvate dehydrogenase (Molecular Probes, Eugene, OR); mouse monoclonal anti-trans-Golgi network38 (Abcam); rat monoclonal anti-ZO-1 (Chemicon, Temecula, CA); and normal rabbit IgG (Santa cruz). Rabbit anti-ERp57 antiserum was prepared against the 17-amino acid C-terminal peptide (VIQEEKPKKKKKAQEDL) of human ERp57 as described [12]. Subfractionation of rat liver mitochondrial outer membrane and intermembrane space All of the procedures were carried out at 4 °C, following the method of Parson et al. [13]. The mitochondrial fraction obtained as described [5] was resuspended in two volumes of 20 mM potassium phosphate buffer, pH 7.4, and allowed to stand at 4 °C for 1 h. The resuspended sample was centrifuged at 3000g for 10 min. The supernatant was centrifuged at 105,000g for 30 min, and the pellet was used as the OM fraction, and the supernatant was used as the IMS fraction. The purity of the mitochondrial OM and IMS was determined by immunoblot analysis with the following antibodies; anti-VDAC antibody for mitochondrial OM, antiadenylate kinase 2 (AK2) for intermembrane space (IMS), antiAIF antibody for inner membrane (IM), pyruvate dehydrogenase (PDH) antibody for matrix, anti-GAPDH antibody for cytosolic fraction, anti-ZO-1 for plasma membrane, anti-calnexin for ER, antitrans-Golgi network-38 for Golgi apparatus as shown (see Ref. [7]). High purities of the mitochondrial OM and IMS were obtained.
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Unbound proteins were eluted with 770 ml of buffer B. The bound proteins were eluted with a linear gradient of 50–300 mM NaCl in buffer A in a total volume of 1.0 L. Four calpain-like activity peaks were detected. The fractions containing calpain-like activity were collected and concentrated by Amicon PM-10 membrane (Millipore Co., Bedford, MA). The concentrates were used for Western blot analyses with anti-l-calpain and anti-m-calpain large subunits, anti-calpain small subunit, anti-ERp57 and antiGrp75 (Fig. 2B). They were also used for immunoprecipitation with anti-l-calpain large subunit, anti-calpain small subunit, and antiERp57 (Fig. 2C). The protein concentration was measured by the Bradford et al. method with bovine serum albumin (BSA) as the standard [14]. We also determined whether that the proteins in the mitochondrial intermembrane space (IMS) were involved in the activation of mitochondrial OM proteins. Fifty-five micrograms of IMS proteins were added to 50 ll of each fraction and incubated at 4 °C for 2 h. Then the calpain activity of each sample was measured (Fig. 3D). Column chromatography of mitochondrial IMS-localized
l- and m-calpains Rat liver mitochondrial IMS were prepared from 30 SD rats (8 weeks-old) as described above. Partial purification of mitochondrial l- and m-calpains was accomplished by DEAE-Sepharose CL-6B column chromatography as we described [5–7] (Fig. 2A). The prepared IMS fraction was dialyzed overnight against 2 L of buffer C (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, and 5 mM 2-mercaptoethanol) containing 50 mM NaCl (buffer D) at 4° C. The dialyzed proteins (200 mg protein) were applied to a DEAE-Sepharose CL-6B column (26.4 400 mm) pre-equilibrated with buffer D at a flow rate of 1.0 ml/min. The fraction size was 10 ml/tube. Aliquots (50 ll) of the fractions were used to assay for calpain activity as described [5]. Unbound proteins, containing mitochondrial m-calpain, were eluted with 700 ml of buffer D. Mitochondrial l-calpain was eluted with a linear gradient of 50– 300 mM NaCl in buffer C in a total volume of 1.1 L. Each fraction was used for the calpain assay in the presence of the OM proteins (Fig. 2B). Fifty micrograms of OM proteins were added to 50 ll of each fraction and incubated at 4 °C for 2 h. In contrast, 1% octylglucoside-solubilized OM proteins were added to each fraction and incubated. Then the calpain activity of each sample was measured as described [5].
Solubilization and column chromatography of mitochondrial OMbound calpains
Partial purification of calpain small subunit in mitochondrial IMS
Mitochondrial OM was prepared from 10 Sprague–Dawley (SD) rats (8 weeks-old) as described above. To prevent the contamination of cytosolic proteins, the mitochondrial surface was treated with proteinase K as described in detail [7]. DEAE-Sepharose CL-6B (Amersham Pharmacia Biotech) column chromatography was used to separate the calpain isoforms in the OM (Fig. 2A). The prepared OMs were suspended in solubilizing buffer (20 mM Tris– HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 5 mM 2-mercaptoethanol, 1% octyl-glucoside, and 50 mM NaCl). The samples solubilized for 30 min at 4 °C and were then centrifuged at 20,000g for 30 min. The supernatants (360 mg protein) were applied to a DEAESepharose CL-6B column (26.4 400 mm) pre-equilibrated with buffer A (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, 5 mM 2-mercaptoethanol, 0.1% octyl-glucoside) containing 50 mM NaCl (buffer B) at a flow rate of 1.0 ml/min. All chromatographic procedures were performed at 4 °C. The amount of each fraction size was 10 ml/tube. Aliquots (50 ll) of the separated fractions were used to assay for calpain activity as described [5].
Sepharose 6B column chromatography (Amersham Pharmacia Biotech) was used to partially purify the calpain small subunit from the mitochondrial IMS (Fig. 4A). Rat liver mitochondrial IMS were prepared from 30 SD rats (8 weeks-old) as described above. The prepared IMS fraction was dialyzed overnight against 2 L of buffer C (20 mM Tris–HCl, pH 7.5, 1 mM EDTA, 1 mM EGTA, and 5 mM 2-mercaptoethanol) containing 140 mM NaCl (buffer E) at 4 °C. The dialyzed proteins (20 mg protein) were applied to a Sepharose column (9 600 mm) pre-equilibrated with buffer E at a flow rate of 0.5 ml/min. The proteins were eluted with buffer E depending on their molecular weight. The molecular weight markers were normal rabbit IgG (140 kDa), BSA (66 kDa), and chymotrypsinogen A (24 kDa). The fraction size was 500 ll/tube. Each fraction was concentrated by Amicon PM-10 membrane (Millipore Co., Bedford, MA). The concentrates were used for Western blot analysis with anti-l- and anti-m-calpain large subunits, anticalpain small subunit, anti-ERp57, and anti-Grp75 antibodies (Fig. 4B).
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Western blotting Western blotting was performed as in our earlier studies [5–7]. After electrophoretic transfer of the proteins to a nitrocellulose membrane, the membranes were treated with the primary antibodies followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit or rabbit anti-goat IgG secondary antibodies (DAKO, Cambridge, UK). The immunoreactive signals were developed with an ECL Western blotting detection kit (Amersham Biosciences) and quantified with a Luminescent Image Analyzer, LAS-3000 (Fujifilm Co., Tokyo, Japan). Assay of ATP concentrations in SD rats liver mitochondrial and cytosolic fractions To determine the concentration of ATP in SD rat liver mitochondrial and cytosolic fractions, we used an ATP measurement kit (Bio Assay Systems, CA). This kit provides a rapid method to measure intracellular cytosolic or organelle ATP. In the presence of luciferase, ATP immediately reacts with the Substrate D-luciferin to produce light. The light intensity is a direct measure of ATP concentration. We measured ATP concentration of SD rat liver, kidney, heart, and brain cytosolic and mitochondrial fractions following a manual method of the kit. The ATP concentration of rat liver mitochondrial and cytosolic fractions were 1.3 and 1.5 mM, respectively. The concentrations of both kidney mitochondrial and cytosolic fractions were 1.1 mM. In heart and brain, ATP concentrations were relatively high (2–4 mM). Effects of calpain small subunit, Grp75, and ATP on calpain activity in mitochondrial OM Mitochondrial OM fractions were prepared from SD rat (8 weeks-old) liver and suspended in buffer E. The samples (100 lg protein/tube) were incubated at 4 °C for 30 min with or without Sepharose 6B-purified calpain small subunit (10 lg protein), Grp75 protein (1 lg protein), 2 mM final concentrations of ATP, TTP, GTP, and CTP. Then the calpain activity of each sample was assayed (Fig. 5A). Effects of Ca2+, ATP, calpain small subunit, and Grp75 on releases of mitochondrial calpains from OM Mitochondrial OM fractions were prepared from SD rat (8 weeks-old) liver, and the prepared OM was suspended in buffer E. The samples (200 lg protein/tube) were incubated at 30 °C for 3 min with or without Sepharose 6B-purified calpain small subunit (10 lg protein), Grp75 protein (1 lg protein), 2 mM final concentrations of ATP, TTP, GTP, CTP, 50 lM Ca2+ or 500 lM Ca2+. Each sample was centrifuged at 100,000g for 1 h and the supernatants were concentrated with Amicon PM-10 membrane. The concentrates were used for Western blot analyses with anti-l-calpain and m-calpain large subunits (Fig. 5B). Ca2+ requirement of m-calpain release from OM SD rat liver mitochondrial OM was suspended in buffer E. The samples (200 lg protein/tube) were incubated at 30 °C for 3 min with Sepharose 6B-purified calpain small subunit (10 lg protein), Grp75 protein (1 lg protein), 2 mM ATP and 50 lM PD150606 in the presence of different concentrations of CaCl2 (0–1000 lM). Each sample was centrifuged at 100,000g for 1 h, and the supernatants were concentrated with Amicon PM-10 membrane. The concentrates were used for Western blot analyses with antim-calpain large subunits (Fig. 5C). Immunoreactive bands were
quantified using a LAS-3000, luminescent image analyzer (Fujifilm Co., Tokyo, Japan). Ca2+ dependency of mitochondrial m-calpain activity The Ca2+ dependency of mitochondrial m-calpain was determined using DEAE-Sepharose-purified IMS m-calpain as described [7] (Fig. 5C). The samples (50 lg protein) were added to 450 ll of assay solution (100 mM Tris–HCl, pH 7.5, 10 mM 2-mercaptoethanol, 100 mM KCl, 1 mM EGTA, 20 lM Succ-Leu-Tyr-AMC) in the presence of different concentrations of CaCl2 (0–1000 lM) and incubated at 25 °C. The continuous methods of calpain assay were performed as described in detail [5]. Immunoprecipitation analyses of Grp75-m-calpain released from OM interaction We also performed immunoprecipitation analyses to determine whether the mitochondrial m-calpain released from OM form complexes with calpain small subunit and Grp75 (Fig. 5D). The prepared OMs (200 lg protein/tube) were incubated at 30 °C for 3 min with Sepharose 6B-purified calpain small subunit (10 lg protein), Grp75 protein (1 lg protein), 2 mM final concentrations of ATP, and 50 mM Ca2+. The samples were centrifuged at 100,000g for 1 h. Polyclonal antibody to Grp75 (1 lg) was incubated with the supernatants containing mitochondrial m-calpain released from the OM in buffer E. Normal rabbit IgG was used as a control for the anti-Grp75 antibody. After standing for 18 h at 4 °C, excess protein G-Sepharose 4 Fast Flow was added (12 ll/ tube), and allowed to stand for 2 h at 4 °C. The preparations were centrifuged at 12,000 rpm for 5 min at 4 °C. The pellets were washed six times with 50 mM Tris–HCl, pH 7.4, containing 140 mM NaCl, and 0.1% Emulphogen BC 720. The co-immunoprecipitated proteins were analyzed by Western blot analyses with anti-m-calpain large subunit and anti-calpain small subunit. Results Activation of mitochondrial OM-bound calpains by different detergents The calpain specific activity of non-solubilized OM was 60 FI/min/mg, and it increased fivefold after solubilization with digitonin, twofold with CHAPS, and threefold with Tween and octylglucoside (Fig. 1). The increased calpain activities were inhibited by 95% by PD150606, a calpain specific inhibitor. Addition of IMS proteins to octyl-glucoside-solubilized OM proteins increased the calpain specific activities by sixfold compared to non-treated IMS fraction (data not shown). This increase in the calpain activity was inhibited by PD150606 by 90%. These results indicated that the IMS proteins activated the OM-bound calpains or the OM proteins activated the IMS calpains. We also examined whether OM proteins activated IM calpain or IM proteins activated OM calpains by mixing each fraction. However, the OM and IM calpain activities were not changed (data not shown). These findings suggested that the activator of OM calpains is not localized in the IM or that of IM calpains is not present in the OM. Effects of OM proteins on mitochondrial l- and m-calpains in mitochondrial IMS We next examined whether the mitochondrial OM proteins were activated the mitochondrial l- and m-calpains of the IMS (Fig. 2). Mitochondrial l- and m-calpains were separated by DEAE-Sepharose CL-6B column chromatography as described [7].
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Fig. 1. Effects of different detergents and IMS proteins on the activation of mitochondrial calpains in OM. Mitochondrial OM proteins were solubilized with a 1% final concentration of sodium deoxycholate, digitonin, CHAPS, Nonidet p-40 (NP40), n-dodecyl-b-D-maltoside (DDM), Triton X-100 (TX-100), Tween and octylglucoside. Each solubilized protein was isolated by centrifugation as described in the Materials and methods section. The samples solubilized with each detergent were assayed for calpain activity. The calpain specific activity of non-solubilized OM is 60 FI/min/mg. The activities increased by about fivefold after solubilization with digitonin, twofold with CHAPS, and threefold with Tween and octyl-glucoside. The increased activities caused by the solubilization with several detergents were inhibited by 95% by the calpain specific inhibitor, PD150606. Data are expressed as means ± SD n = 3.
We obtained three peaks of calpain activity; an unbound protein fractions (Peak 1), a peak at 120 mM NaCl (Peak 2), and a peak at 150 mM NaCl (Peak 3; Fig 2A). We found that Peak 1 contained mitochondrial m-calpain, Peak 2 contained mitochondrial l-calpain, and Peak 3 contained ERp57-associated mitochondrial l-calpain [7]. Unsolubilized OM proteins were added to each fraction but no further activation of mitochondrial l- and m-calpains was found (Fig. 2B). These results demonstrated that mitochondrial OM proteins do not affect the specific activities of mitochondrial l- and m-calpains in the IMS. In addition, these data suggested that the l- and m-calpains located in the IMS do not affect the activity of OM-bound calpain. In contrast, the addition of octylglucoside-solubilized OM proteins increased the calpain activities and a new peak appeared (Fig. 2B). We detected the calpain small subunit in the new peak by Western blot analysis (data not shown). We suggest that the calpain small subunit in IMS activate octyl-glucoside-solubilized OM-bound calpains.
Identification of calpain isoforms in mitochondrial OM We separated the mitochondrial OM-bound calpains by DEAESepharose CL-6B column chromatography. We solubilized the OM proteins with 1% octyl-glucoside and applied it to the column. The unbound proteins were eluted with buffer B, and the adsorbed proteins were eluted with a linear gradient of 50–300 mM NaCl in buffer A (Fig. 3A). We detected four peaks of calpain activity; an unbound protein fraction (Peak 1), a peak for 100 mM NaCl (Peak 2), a peak for 125 mM NaCl (Peak 3), and a peak for 200 mM NaCl (Peak 4). Each peak was collected and concentrated. Western blot analyses showed that the autolyzed forms of l-calpain large subunit and calpain small subunit were contained in Peak 1, and the intact forms of l-calpain and m-calpain large subunits in Peak 4 (Fig. 3B). Interestingly, the calpain small subunit and Grp75 were not detected in the fraction containing l-calpain and m-calpain large subunits. We detected mitochondrial calpain-associated chaperon ERp57 in Peaks 1, 3, and 4. Grp75, a protein associated with mitochondrial m-calpain, was present in Peak 3. We obtained calpain-like protease activities in Peaks 2 and 3 as shown in Fig. 3A, although no calpain large and small subunits
Fig. 2. Effects of OM proteins on mitochondrial l- and m-calpains in IMS. (A) Mitochondrial l- and m-calpains were separated by DEAE-Sepharose CL-6B column chromatography using the IMS fraction as described in Materials and methods section. Three peaks of calpain activity can be seen: an unbound protein fractions (Peak 1), a peak at 120 mM NaCl, and a peak at 150 mM NaCl (Peak 3). Peak 1 contains the Grp75-associated mitochondrial m-calpain, Peak 2 contains mitochondrial l-calpain, and Peak 3 contains ERp57-associated mitochondrial lcalpain. (B) Unsolubilized OM proteins and 1% octyl-glucoside-solubilized OM proteins were added to each fraction and incubated at 4 °C for 2 h. Details are described in the Materials and methods section. No significant increase in the calpain activity is observed in unsolubilized OM proteins-added fractions. Addition of octyl-glucoside-solubilized OM proteins increases the calpain activities and a new peak appeared. Results are representative of three independent experiments (A and B).
were detected as shown in Fig. 3B. So we checked the activities of Peaks 2 and 3 with calpain specific inhibitor, PD150606, and we found that PD150606 did not inhibit the activities (data not shown). In addition, we found that the activities of Peaks 2 and 3 were obtained in the absence of Ca2+. The calpain substrate we used in the assay, Suc-Leu-Tyr-AMC, was also cleaved by cathepsins to some degree [15]. Therefore, these results suggested that Peaks 2 and 3 contained cathepsin-like proteases. Immunoprecipitation analysis showed that the 55–65 kDa autolyzed l-calpain large subunit did not associate with the 30 kDa calpain small subunit in Peak 1, and the 80 kDa l-calpain large subunit bound to ERp57 in Peak 4 (Fig. 3C). We also examined whether the mitochondrial IMS proteins activated the OM-bound calpains. The calpains of peak 4 containing l- and m-calpain large subunits were significantly activated by IMS proteins (Fig. 3D).
Presence of calpain small subunit in mitochondrial IMS Earlier, we found an unknown calpain-associated small subunit or free small subunit in the IMS. We expected that the free calpain small subunit was involved in the activation of the OM-bound calpains. We confirmed that the free calpain small subunit was
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Fig. 3. Identification of calpain isoforms in mitochondrial OM and their activations with IMS proteins. (A) The OM proteins were separated with DEAE-Sepharose CL-6B column chromatography. Details are described in the Materials and methods section. The octyl-glucoside-solubilized OM proteins were applied to the column. Four peaks of calpain-like activity are present; an unbound protein fractions (Peak 1), at 100 mM NaCl (Peak 2), at 125 mM NaCl (Peak 3), and at 200 mM NaCl (Peak 4). (B) Western blot analyses of each peak of calpain-like activity (30 lg protein/lane). From top to bottom panel, antibodies used were anti-l-calpain large subunit, m-calpain large subunit, calpain small subunit, ERp57, and Grp75 antibodies. The IMS fraction (20 lg protein) was used as controls of ERp57 and Grp75 (far right lane). Peak 1 contains autolyzed lcalpain large subunit, calpain small subunit and ERp57. Peak 3 contains ERp57 and Grp75. Peak 4 contains the intact l-calpain, m-calpain large subunit and ERp57 but not calpain small subunit. (C) Immunoprecipitation analyses of mitochondrial l-calpain with anti-calpain small subunit and anti-ERp57 antibodies. Details are described in the Materials and methods section. The 55–65 kDa autolyzed l-calpain large subunit did not associate with the 30 kDa calpain small subunit in Peak 1, and the 80 kDa l-calpain large subunit bound to the ERp57 in Peak 4. Results are representative of three independent experiments. (D) Effects of IMS proteins on each peak. Details are described in the Materials and methods section. In the presence of IMS proteins, only Peak 4, which contains l-calpain and m-calpain large subunits, is activated twofold to non-treated sample. Results are representative of three independent experiments.
present in the IMS by Sepharose 6B gel filtration column chromatography (Fig. 4A). Each fraction was concentrated by Amicon PM10 membrane and used for Western blot analyses of the calpain small subunit. The calpain small subunit was contained in the >140 kDa, 110 kDa, and 24–30 kDa protein fractions (Fig. 4B). The >140 kDa protein fraction contained ERp57-associated lcalpain and Grp75-associated mitochondrial m-calpains, while the 110 kDa protein fraction contained l-calpain. ERp57 and Grp75 were detected in different fractions. We detected the small subunit in the fractions that did not contain calpain activities, and the large subunits of l- and m-calpains in the 24–30 kDa protein fractions. These results indicated that part of the calpain small subunits is free in the IMS. To confirm the effects of the free calpain small subunits on the activity of the OM-bound calpains, we added the Sepharose 6B-purified calpain small subunit to octyl-glucoside-solubilized OM proteins and incubated the mixture at 4 °C for 2 h. The calpain small subunit significantly activated the OM-bound calpains (Fig. 4C). The calpain small subunit that had been exposed to anti-calpain small subunit did not activate the OM-bound calpains, and the boiled small subunit also did not activate the OM-bound calpains. These results showed that the free calpain small subunit modulated the activity of the OM-bound calpains. Effects of calpain small subunit, Grp75, and ATP on calpain activity in mitochondrial OM The results shown in Figs. 1–4 suggested that the free calpain small subunit can activate solubilized OM-bound calpains. However, the results obtained on solubilized OM do not necessarily
reflect its activity in the mitochondria. To examine this, we used unsolubilized OM and observed the effects of calpain small subunit, Grp75, and ATP on the calpain activities in the unsolubilized OM. We added free calpain small subunit, Grp75, ATP, GTP, CTP, and TTP to the unsolubilized OM under different conditions. The samples were incubated at 4 °C for 30 min, and the calpain activity was assayed. The calpain specific activity of non-solubilized OM was 60 FI/min/mg (Fig. 5A). The activity was twice as high as the non-treated OM in the presence of calpain small subunit. Grp75 did not affect the activity, but the addition of the small subunit and Grp75 increased the activity by about threefold compared with the non-treated condition. In addition, we detected an increase in the calpain activity by fivefold over non-treated OM in the presence of small subunit, Grp75, and ATP. However, it appeared that the calpain activity in the OM remained unaffected by the presence of TTP, GTP, and CTP. Even if the Grp75 protein was not added to the OM, the activity increased threefold over the non-treated one in the presence of the small subunit and ATP. These data indicated that the OM-bound calpain large subunits were activated by Grp75 and ATP as well as the calpain small subunit. Effects of Ca2+, ATP, calpain small subunit, and Grp75, and ATP on releases of mitochondrial calpains from OM The mitochondrial unsolubilized OM-bound calpains were activated by Grp75, calpain small subunit, and ATP (Fig. 5A). It was not certain whether mitochondrial calpains are activated on the OM or after being released from the OM. Therefore, we examined whether the OM-bound m-calpain was released from the OM and then
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activated (Fig. 5B). We added the 50 lM Ca2+, 500 lM Ca2+, ATP, the free calpain small subunit, and Grp75 to the unsolubilized OM in different combinations. The samples were then incubated at 30 °C for 3 min and centrifuged at 100,000g for 1 h to obtain the proteins released from the OM. The supernatants containing the proteins released from the OM were analyzed by Western blotting for l-calpain and m-calpain large subunits. Mitochondrial m-calpain large subunit was released from the OM in the presence of 50 lM Ca2+, ATP, the calpain small subunit, and Grp75. High concentration of Ca2+ (500 lM Ca2+) induced the release of the autolytic products of m-calpain large subunit as well as the intact forms. Without Ca2+, mitochondrial m-calpain large subunit was minimally released. The release of m-calpain large subunit was weakly inhibited without ATP. The release was blocked by the absence of the calpain small subunit and Grp75. Addition of 50 lM Ca2+ alone induced a slight release of m-calpain large subunit. ATP alone, small subunit alone, or Grp75 alone did not induce the release. On the other hand, the l-calpain large subunits were not released from the OM under all conditions. Ca2+ requirement of m-calpain releases from OM We examined the effects of Ca2+ concentrations on the releases of the m-calpain large subunit from the OM by Western blotting (Fig. 5C). Immunoreactive bands of released m-calpain large subunit were quantified using a luminescent image analyzer. The m-calpain large subunits began to be released from the OM at 25 lM Ca2+, and 40% of these were released at 50 lM Ca2+. However, almost all IMS m-calpain was not activated at 25–100 lM Ca2+. These results suggested that the release occurred before m-calpain was activated. Immunoprecipitation analyses of Grp75-m-calpain released from OM interaction Immunoprecipitation analyses showed that the m-calpain large subunit released from the OM formed complexes with the calpain small subunit and Grp75 (Fig. 5D). Discussion
Fig. 4. Presence of a free calpain small subunit in IMS. Details are described in Materials and methods section. (A) Sepharose 6B column chromatography of mitochondrial IMS proteins. Molecular weight markers used were normal rabbit IgG (140 kDa), BSA (66 kDa) and chymotrypsinogen A (24 kDa). (B) Western blot analysis of l- and m-calpain large subunits, calpain small subunit, ERp57 and Grp75 using Sepharose 6B-separated IMS fractions (20 lg protein/lane). We detected the calpain small subunit in the >140, 110, and 24–30 kDa protein fractions. The circled fractions in panel A contain the calpain small subunit. The >140 kDa protein fraction contains ERp57-associated l-calpain and Grp75-associated mitochondrial m-calpains, and the 110 kDa protein fraction contains l-calpain. The calpain small subunit was also detected in the 24–30 kDa protein fractions. These results indicate that a part of calpain small subunits are free in the IMS. (C) Effects of the free calpain small subunit on the activity of mitochondrial OM-bound calpains. The Sepharose 6B-purified calpain small subunit was incubated with octylglucoside-solubilized OM proteins at 4 °C for 2 h. The calpain small subunit significantly activated the OM-bound calpains (250% compared to the non-treated OM proteins). Absorption of calpain small subunit with anti-calpain small subunit antibody does not activate the OM-bound calpains. The partial purified calpain small subunit was boiled at 80 °C for 3 min and incubated with OM proteins. An increase of calpain activity by the boiled small subunit was not detected. Data are expressed as means ± SD, n = 3, ⁄P < 0.01 vs the calpain specific activity of nontreated condition.
Our results showed that IMS-localized free calpain small subunit and Grp75 play critical roles for activating OM-bound m-calpain. They also showed that pre-activated mitochondrial m-calpain large subunit is released from the OM together with the calpain small subunit and Grp75 by exposure to low concentrations of Ca 2+. After their releases from the OM, mitochondrial m-calpain appeared to be further activated by the increased mitochondrial Ca2+ level. We previously suggested that OM-bound calpains existed as the pre-active forms under physiological condition [5]. This is probably because the OM-bound mitochondrial l- and m-calpains are not bound to the calpain small subunit (Fig. 3). The mitochondrial m-calpain domain IV, which is associated with the calpain small subunit, is probably embedded in the OM. This would then suggest that the OM-bound mitochondrial m-calpain large subunit does not bind to the small subunit and has weak activity. The OM-bound calpains are activated by several detergents such as digitonin, Tween, and octyl-glucoside (Fig. 1). This indicates that the solubilization of OM-bound calpains allows the calpains to change their conformation from the pre-active to the active form. In the case of cytosolic m-calpain, the small subunit plays an essential role in the conformational change to the active form [16,17]. The restriction of the conformational changes of the
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Fig. 5. Effects of Ca2+, calpain small subunit, Grp75 and ATP on calpain activity of mitochondrial OM-bound calpains and their releases from OM. Details are described in Materials and methods section. (A) Effects of calpain small subunit, Grp75, ATP, TTP, GTP, and CTP on the activity of mitochondrial OM-bound calpains. The calpain specific activity of non-solubilized OM was 60 FI/min/mg (column 1). The calpain small subunit activated the OM-bound calpains twice as high as the non-treated OM (column 2). The activity is not changed by the presence of Grp75 (column 3). The addition of calpain small subunit and Grp75 induces an increase in the activity by about threefold of the non-treated one (column 4). Further addition of ATP activated the OM-bound calpains over sixfold as high as non-treated one (column 5). TTP, GTP, and CTP had no significant effects for the OM-bound calpain activities (columns 6–8). The OM-bound calpains were not activated in the presence of only ATP (column 9). The addition of calpain small subunit and ATP induced the activation about threefold of the non-treated one (column 10). Without calpain small subunit, the OM-bound calpains are not activated (column 11). All data are mean ± SD, n = 3, ⁄P < 0.01, ⁄P < 0.001 vs the calpain specific activity of non-treated OM. (B) Effects of Ca2+, ATP, calpain small subunit, and Grp75 on releases of mitochondrial calpains from OM. Mitochondrial m-calpain large subunit was released from the OM in the presence of 50 lM Ca2+, ATP, calpain small subunit, and Grp75 (lane 1). High concentration of Ca2+ (500 lM) induced the releases of the intact and autolyzed mitochondrial m-calpain large subunit in the presence of ATP, calpain small subunit, and Grp75 (lane 2). Mitochondrial m-calpain large subunit was not released from the OM without Ca2+ (lane 3). Without ATP, their releases weakened slightly (lane 4). The releases were strongly blocked without calpain small subunit (lane 5) and Grp75 (lane 6). Addition of 50 lM Ca2+ alone induced a slight release of m-calpain large subunit (lane 7). ATP alone, small subunit alone, or Grp75 alone did not induce the release (lane 8–10). In contrast, mitochondrial l-calpain large subunit was not released from the OM under all conditions (lower panel). Mitochondrial OM was used for the positive controls of l-calpain and m-calpain large subunits (lane 11, 40 lg protein). Results are representative of three independent experiments. (C) Ca2+ requirements for the release of m-calpain from the OM and Ca2+ dependency of mitochondrial m-calpain activities. We examined the correlation between Ca2+ requirement for m-calpain releases from the OM and Ca2+ dependency of mitochondrial m-calpain activities. The m-calpain large subunits begin to be released from the OM at 25 lM Ca2+, which increases to 40% at 50 lM Ca2+. However, almost all IMS m-calpain was not activated at 25–100 lM Ca2+. Data are the means ± SD, n = 3. (D) Immunoprecipitation of mitochondrial m-calpain released from OM with anti-Grp75 antibody. Both mitochondrial m-calpain large (open arrowhead) and small subunits (solid arrowhead) are co-precipitated with Grp75. The results show that mitochondrial m-calpain large subunits released from the OM formed the complexes with calpain small subunits and Grp75.
OM-bound m-calpain large subunit is manifested by the weak activity of OM-bound calpains. Another possibility is that permeabilization of OM proteins dissociated the OM-bound calpains from their binding partners such as the calpastatin-like inhibitory proteins. At present, we are trying to identify the binding partners by column chromatography, immunoprecipitation with anti-l- and m-calpain large subunits antibodies, and LC–MS/MS analyses. We found that Ca2+ and the free calpain small subunit release the mitochondrial m-calpain large subunit from the OM and activate it (Figs. 4 and 5). The release occurred at a low concentration of Ca2+, which does not activate the mitochondrial m-calpain (Fig. 5B and C), and the released mitochondrial m-calpain combines with the small subunit (Fig. 5D). In contrast, a high concentration of Ca2+ (500 lM Ca2+) causes the autolysis of mitochondrial m-calpain, indicating that it is activated (Fig. 5B). We suggest that the low concentrations of Ca2+ induced conformational changes of the OM-bound m-calpain large subunit but stops short of its activation. It is likely that the conformational change exposes its domain IV to the IMS and leads to the binding of the free calpain small subunit. This heterodimer formation may induce its release from the OM into the IMS. Additional Ca2+ influx would activate
the mitochondrial m-calpain released from the OM. Another important change induced by Ca2+ was the conformational change of the free calpain small subunit in the IMS. The calpain small subunit has Ca2+ binding sites in its domain VI [1]. The conformational change of the free calpain small subunit may increase its accessibility of the subunits to OM-bound m-calpain large subunit to form the heterodimer. Not only Ca2+ and the free calpain small subunit but also Grp75 play significant roles in the release and activation of OM-bound mitochondrial m-calpain large subunit (Fig. 5). Grp75 plays a role in the import into the mitochondria and in the proper folding of nuclear encoded proteins in an ATP-dependent manner [18,19]. We previously found that m-calpain bound to Grp75 in mitochondrial IMS [7]. In contrast, the present study showed that the mcalpain large subunit does not bind to both small subunit and Grp75 in the OM. We demonstrated that the molecular forms of calpains are mostly different between IMS and OM. The OMlocation of l- and m-calpains under imperfect conditions such as l-calpain large subunit/ERp57 and m-calpain large subunit alone indicates that the OM is mitochondrial calpains settlement until they are supplied to the IMS and are activated. The present study
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indicated that Grp75 promotes the release and activation of OM-bound mitochondrial m-calpain. We suggested that a portion of OM-bound m-calpain large subunit is unfolded, and Grp75 binds to the subunits and makes it properly folded. It is unclear whether Grp75 binds to the OM-bound m-calpain large subunit and then the calpain small subunit associates the calpain. However, this proper folding seems to stimulate the release of mitochondrial m-calpain from the OM. Mitochondrial m-calpain was not released from the OM without Ca2+ in the presence of the calpain small subunit, Grp75, and ATP (Fig. 5B). These findings suggest that Ca2+ causes conformational changes of the OM-bound m-calpain large subunits and then Grp75 chaperones bind to the subunits and pull them into the IMS together with the small subunits. Our results demonstrated that the activation of OM-bound mitochondrial m-calpain occurs in several steps: (1) an apoptotic stimulus leads to the influx of Ca2+ into the cytosol; (2) the prolonged increase of the intracellular Ca2+ level leads to mitochondrial uptake of Ca2+; (3) conformational changes of mitochondrial m-calpain large subunit in the OM occur in response to the Ca2+; (4) the conformational changes lead to the binding of calpain small subunit and Grp75 to OM-bound mitochondrial m-calpain large subunit; (5) this complex formation causes the release of the mitochondrial m-calpain from the OM into the IMS; and (6) a further increase of mitochondrial Ca2+ level activates the mitochondrial m-calpain. The fundamental finding of this study was that OM-bound mitochondrial m-calpains are released to the IMS with the calpain small subunit and Grp75 by influx of Ca2+. We previously suggested that mitochondrial m-calpain in the IMS promotes an apoptotic cell death by releasing tAIF into the cytosol through VDAC–Bax complexes [7]. The increase of mitochondrial m-calpain released from OM must facilitate apoptosis by releasing of tAIF into the cytosol. The present study provides a new insight that the specific inhibition of mitochondrial m-calpain has a therapeutic
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