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DNA-binding proteins of mammalian mitochondria
Mitochondrion 5 (2005) 35–44 www.elsevier.com/locate/mito
DNA-binding proteins of mammalian mitochondria Michael P. Kutsyi*, Natalia A. Gouliaeva, Elena A. Kuznetsova, Azhub I. Gaziev Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290, Russia Received 15 June 2004; received in revised form 1 September 2004; accepted 9 September 2004
Abstract Acid-soluble proteins were isolated from liver and spleen mitochondria and their ability to form complexes with DNA was investigated. According to electrophoresis data, acid-soluble proteins include about 20 polypeptides ranging in the molecular mass from 10 to 120 kDa. It was found that acid-soluble proteins form stable DNA–protein complexes at a physiological NaCl concentration. Different polypeptides possess different degrees of DNA affinity. There is no significant difference between DNA-binding proteins of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In the presence of 5 mg of DNA most polypeptides were bound to DNA, and further increase in DNA amount affected little the binding of proteins to DNA. There was no distinct difference in DNA–protein complex formation of liver mitochondrial acid-soluble proteins with nDNA or mtDNA. Also, it was detected that with these mitochondrial acid-soluble proteins, proteases that specifically cleave these proteins are associated. It was shown for the first time that these proteases are activated by DNA. DNA-binding proteins including DNA-activated mitochondrial proteases are likely to participate in the regulation of the structural organization and functional activity of mitochondrial DNA. q 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: Mitochondria; DNA-binding proteins; Acid-soluble proteins; Histones; Proteases
1. Introduction In mammalian somatic cells, the mitochondrial DNA (mtDNA) is represented by numerous copies (from 1 to several thousands) of double-stranded circular molecules of 16.5 kb (Anderson et al., 1981; Bibb et al., 1981; Takamatsu et al., 2002). DNA in mitochondria is mostly associated with the inner membrane (Adams et al., 1980; Potter et al., 1980). The mtDNA interacts with proteins of the inner
* Corresponding author. Fax: C7 967 330553. E-mail address: [email protected] (M.P. Kutsyi).
mitochondrial membrane via particular D-loop regions (Potter et al., 1980). MtDNA compared to nuclear DNA (nDNA) is more vulnerable to reactive oxygen species (ROS) formed in the mitochondrial respiratory chain. The number of oxidized bases in mtDNA is about 16 times as high as in nDNA (Richter et al., 1988; Richter, 1992). Mutations and deletions in mtDNA may be a cause of aging and development of degenerative diseases in the organism (Wallace, 1992; Cortopassi et al., 1992). It has been suggested that a major contributor to the high sensitivity of mtDNA to damaging ROS influences is the absence of histones in mitochondria. Therefore, in intact mitochondria DNA is not covered by histones and other proteins, as it is
1567-7249/$ - see front matter q 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2004.09.002
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the case in the nuclei (Richter, 1992; Wallace, 1992; Higuchi and Linn, 1995). At the same time, some authors have shown the presence of mtDNA–protein complexes, in which protein is tightly associated with DNA and this linkage is stable to the action of ionic detergents and high concentrations of NaCl. In particular, DNA–protein complexes have been isolated from rat liver mitochondria after their lysis with sarkosyl (Van Tuyle and Kalf, 1972) or with BRIJ 58 in the presence of sodium deoxycholate and 0.5 M NaCl (Van Tuyle and McPherson, 1979). According to electron microscopy data, mtDNA molecules are organized as rosette-like structures with a dense central core (Van Tuyle and McPherson, 1979). They are maintained in the highly folded state by associated proteins. DNA–protein complexes have also been isolated from the yeast S. cerevisiae mitochondria (Rickwood et al., 1981; Miyakawa et al., 1987). From liver mitochondria, a histone-like protein of 16 kDa molecular mass has been isolated which interacts with nucleotide sequences in the region of the D-loop of mtDNA (Pavco and Van Tuyle, 1985). Histone-like proteins have also been obtained from the mitochondria of heart (Yamada et al., 1991), yeast (Caron et al., 1979), and Xenopus laevis (Mignotte et al., 1985). Recently it has been shown that multiple mtDNA molecules are organized into discrete DNA–protein complexes called ‘nucleoids’ (Miyakawa et al., 1987; Garrido et al., 2003). At the present time, the protein composition of mitochonrial nucleoids and the mechanisms by which mtDNA functions within this dynamic DNA–protein structure have not yet been completely elucidated. It has been shown that nucleoids from the mitochondria of primary human fibroblast cells, 143B osteosarcoma and A549 adenocarcinoma cells contain mitochondrial gamma-polymerase, the transcriptional factor A of mitochondria, and mitochondrial single-stranded DNA binding proteins (Garrido et al., 2003). Probably, proteins loosely bound to DNA also participate in the packing of mtDNA within the nucleoid. When DNA or DNA–protein complexes are isolated under rigorous conditions with the use of ionic detergents or high salt concentrations, the proteins loosely bound to DNA dissociate and hence are not detected in isolated preparations of DNA and DNA–protein complexes. Recently, proteins binding to RNA have been shown
to be present in mammalian mitochondria (Koc and Spremulli, 2003). In the present work, a spectrum of acid-soluble DNA-binding proteins of rat liver and spleen mitochondria is presented and their ability to form complexes with DNA is shown. Also it is shown that these acid-soluble proteins contain proteases including DNA-activated ones.
2. Materials and methods 2.1. Materials All chemical reagents were of analytical grade. All reagents for polyacrylamide gel electrophoresis (PAGE), calf thymus DNA, DNaase, RNaase A, antipain, leupeptin, phenylmethylsulphonyl fluoride (PMSF), N-ethymaleimide (NEM), and digitonin were obtained from Sigma Chemical Company. Proteinase K and sodium dodecyl sulfate (SDS) were obtained from AppliChem GmbH (Germany), lambda DNA from phage isolated from the E. coli W3110 (c/1857 Sam7) strain, protein molecular mass standards (b-galactosidase, 116.0 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45.0 kDa; lactate dehydrogenase, 35.0 kDa; restriction endonuclease Bsp981, 25.0 kDa; b-lactoglobulin, 18.4 kDa; lysozyme, 14.4 kDa) were obtained from Fermentas MBI. 2.2. Isolation of nuclei and mitochondria Male Wistar rats weighing 180–200 g were used. Rat liver and spleen mitochondria were isolated by differential centrifugation, as described by Pedersen et al. (1978). Mitochondria were purified from lysosomal impurities by treatment with digitonin (Loewenstein et al., 1970). Nuclei from liver and spleen were isolated as described by Blobel and Potter (1966). DNA from liver mitochondria was isolated according to Suter and Richter (1999). 2.3. Extraction of acid-soluble proteins from nuclei and mitochondria Acid-soluble proteins (histones) were extracted from liver and spleen nuclei with 0.2 M H2SO4, as described by Bonner et al. (1968). Histones thus
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obtained were precipitated with ethanol and extracted once again with 0.2 M H2SO4. Analogously, acidsoluble proteins were extracted from liver and spleen mitochondria. Protein concentration was determined according to Lowry et al. (1951). 2.4. Electrophoresis of proteins in polyacrylamide gel Electrophoresis of acid-soluble proteins was performed in 15% SDS-polyacrylamide gel by the method of Laemmly (1970). Enzyme-electrophoresis of proteases associated with acid-soluble proteins of mitochondria was carried out in 15% SDS-polyacrylamide gel containing covalently bound casein according to Kelleher and Juliano (1984) with minor modification. Acid-soluble mitochondrial proteins were dissolved in a buffer containing SDS but with no 2-mercaptoethanol. Samples were not boiled before being applied to gel. After electrophoresis, the gel was washed for 1.5 h with 0.02 M Tris–HCl buffer, pH 8.0 containing 0.5% Triton X-100 to remove SDS. In order to detect protease activity, the gel was incubated for 48 h at 37 8C in 0.02 M Tris– HCl buffer, pH 8.0, containing 25 mM NaCl. After incubation, the gel was stained with Coomassie brilliant blue (CBB R-250). 2.5. Formation of DNA–protein complexes To obtain DNA–protein complexes, different amounts of DNA (2, 5, and 10 mg) were added to 50 mg of acid-soluble mitochondrial proteins in 0.02 M Tris–HCl, pH 7.4, containing 0.15 M NaCl. In order to study the effect of NaCl on the formation of DNA–protein complexes, 0.15–0.5 M NaCl was added to 50 mg of protein and 10 mg of DNA. Thirty minutes later, DNA–protein complexes were sedimented by centrifugation (5000!g, 30 min at 4 8C). Then the pellet and supernatant were subjected to electrophoresis in 15% SDS-polyacrylamide gel. The formation of DNA–protein complexes was also judged from comparing the binding of actinomycin D to free DNA and DNA complexed with protein (Muller and Crothers, 1968). In the case of DNA–protein complex formation, the binding of actinomycin D must be lower than in the case of free DNA.
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2.6. Assay of protease activity In order to determine the activity of proteases associated with acid-soluble mitochondrial proteins, 50 mg of proteins in 0.04 M Tris–HCl buffer, pH 8.0, were incubated for 1.5–3.0 h at 37 8C in the presence or in the absence of 10 mg of native or denatured DNA. DNA was denatured by heating for 10 min at 100 8C and then rapidly cooled. After incubation, samples were subjected to electrophoresis in 15% SDS-polyacrylamide gel. After staining with Coomassie brilliant blue, gels were scanned with subsequent computation. Protease activity was judged from the amount of polypeptides degraded in the course of incubation. In estimating the pH dependence of the activity of proteases associated with mitochondrial acid-soluble proteins, these proteins were incubated for 3 h at 37 8C and the amount of degraded proteins was determined by the method of Lowry et al. (1951).
3. Results 3.1. DNA-binding acid-soluble proteins of mitochondria Acid-soluble proteins were isolated from mitochondria and nuclei of rat liver and spleen by extraction with 0.2 M H2SO4. It should be noted that extraction with 0.2 M H2SO4 is commonly used to isolate basic proteins, histones, from the nuclei (Bonner et al., 1968). As is seen from electrophoregrams in Fig. 1 (lanes 3, 5), the basic proteins from liver and spleen nuclei are represented by five histone fractions, which is consistent with the literature data (Bonner et al., 1968; Hnilica, 1975; Olszewska and Tait, 1980). However, in our experiments the treatment of liver and spleen mitochondria with 0.2 M H2SO4 also gave acid-soluble proteins. Upon electrophoresis in 15% SDS-polyacrylamide gel, these proteins were divided into more than 20 polypeptides with molecular masses of 10–120 kDa. The electrophoretical spectra of acid-soluble proteins from spleen mitochondria are similar to those from liver mitochondria, although there are distinct differences in the ratios between particular peptides (Fig. 1, lanes 2, 4). As upon the treatment of nuclei with sulfuric acid the basic
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Fig. 1. Acid-soluble proteins of mitochondria and nuclei from rat liver and spleen. Lane 1, protein markers; Lane 2, liver mitochondrial proteins; Lane 3, liver nuclear proteins (histones); Lane 4, spleen mitochondrial proteins; Lane 5, spleen nuclear proteins (histones).
proteins, histones, are extracted, it can be assumed that the acid-soluble proteins extracted by the same procedure from mitochondria may mostly possess the properties of basic proteins. 3.2. Formation of nucleoproteid complexes: acid-soluble proteins–DNA In further experiments, it was found that acidsoluble proteins from liver and spleen mitochondria form nucleoproteid complexes with DNA at the physiological NaCl concentration. These complexes are easily isolated by centrifugation from the DNA and proteins that are unable to form complexes and remain in the supernatant (Fig. 2). There is no significant difference between DNA-binding proteins
Fig. 2. Formation of mitochondrial acid-soluble proteins–DNA complexes. MtDNA (10 mg) or nDNA (10 mg) were added to 50 mg of spleen mitochondrial proteins in 0.02 M Tris–HCl buffer, pH 7.4, containing 0.15 M NaCl. After centrifugation, the proteins of pellet (P) and those of supernatant (S) were subjected to electrophoresis. Lane 1; initial proteins (50 mg). Lanes 2 and 3; protein and mtDNA. Lanes 4 and 5; protein and nDNA.
of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In studying the dependence of DNA–protein complex formation on the amount of nDNA or mtDNA, it was found that addition of 2 mg of DNA to the reaction mixture resulted in the binding of substantial amounts of mitochondrial acid-soluble proteins (Fig. 3). In the presence of 5 mg of DNA most polypeptides were bound to DNA, and further increase of DNA amount to 10 mg influenced little the binding of proteins to DNA. There was no distinct difference in DNA–protein complex formation of liver mitochondrial acid soluble proteins with nDNA or mtDNA (Fig. 3). The study of the NaCl concentration dependence of the binding of liver
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Fig. 3. Dependence of acid-soluble protein–DNA complex formation on DNA concentration. MtDNA or nDNA (2, 5, 10 mg) were added to 50 mg of liver mitochondrial proteins in 0.02 M Tris–HCl buffer, pH 7.4, containing 0.15 M NaCl. After centrifugation, the proteins of pellet (P) and those of supernatant (S) were subjected to electrophoresis. (A) mtDNA–protein complexes; (B) nDNA–protein complexes. Lane 1; initial proteins (50 mg). Lanes 2 and 3; proteins and 2 mg DNA. Lanes 4 and 5; proteins and 5 mg DNA. Lanes 6 and 7; proteins and 10 mg DNA.
mitochondrial acid-soluble proteins to DNA showed that when NaCl concentration increases, the formation of nucleoproteid complexes gradually decreases to stop completely at 0.5 M NaCl (Fig. 4). The formation of DNA–protein complexes was also determined from the capacity of proteins to compete with actinomycin D for DNA-binding sites. The binding of actinomycin D to DNA was known to be accompanied by a change in its optical properties (Muller and Crothers, 1968). In the formation of a DNA–protein complex, the binding of actinomycin D must be lower for the DNA–protein complex than for free DNA. The absorption spectra of free actinomycin D, the nDNA-actinomycin D complex and a reconstructed nucleoproteid–actinomycin D complex are presented in Fig. 5A. It appeared that addition of acidsoluble proteins from liver mitochondria to nDNA at the ratio 1.5:1 reduced actinomycin binding by about 40%, which points to the formation of a DNA–protein
complex. When lDNA was used, a nearly the same spectrophotometric characteristics of the DNA–protein complex was observed (Fig. 5B). Lambda DNA from the phage isolated from the E. coli W3110 (c/1857 Sam7) strain consists of 48502 bp, it is nearly 3 times as long as mitochondrial DNA. The study of the differential spectra of DNA-actinomycin D and DNA–protein-actinomycin D against actinomycin D (Fig. 6) showed changes in the character of the spectral curve for the complex DNA–protein-actinomycin D as compared to the DNA-actinomycin D complex. These differences are due to different amounts of actinomycin D bound to free DNA and the DNA–protein complex. The differential spectra presented show distinctly that to DNA complexed with protein a substantially less amount of actinomycin D is bound than to free DNA since the binding site on DNA in the DNA–protein complex are partially occupied by protein. Addition of NaCl to
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a concentration of 1 M resulted in dissociation of the DNA–protein complex, which is indicated by a change in the spectral curve (it tends to became a direct line). Thus, the results point to that acid-soluble mitochondrial proteins form complexes with DNA. 3.3. Proteases associated with acid-soluble proteins of mitochondria. DNA-activated proteases
Fig. 4. Dependence of acid-soluble protein–DNA complex formation on NaCl concentration. NaCl (0.15 M, 0.35 M, 0.42 M, 0.5 M) and 10 mg nDNA were added to 50 mg of liver mitochondrial proteins in 0.02 M Tris–HCl buffer, pH 7.4. After centrifugation, proteins of the pellet (P) and those of the supernatant (S) were subjected to electrophoresis. Lane 1; initial proteins (50 mg). Lanes 2 and 3; proteins and nDNA with 0.15 M NaCl. Lanes 4 and 5; proteins and nDNA with 0.35 M NaCl. Lanes 6 and 7; proteins and nDNA with 0.42 M NaCl. Lanes 8 and 9; proteins and nDNA with 0.5 M NaCl.
It has been well known that mitochondria contain many proteases involved in the metabolism of mitochondrial proteins (Bota and Davies, 2001, review). In our investigation it was revealed that along with acid-soluble proteins, also proteases were extracted that cleaved various polypeptides of these proteins at various intensity (Fig. 7). We showed for the first time that the activity of these proteases increased in the presence of DNA and that denatured nDNA and mtDNA activated them to a higher degree than native nDNA and mtDNA do (Fig. 7). The intensity of cleavage of the acid-soluble proteins by these proteases was higher than the cleavage of other mitochondrial proteins. The optimum activity of proteases associated with acid-soluble proteins was observed at pH 8.0 (data not shown). When acid-soluble proteins from liver mitochondria were incubated with the protease inhibitors antipain, leupeptin, PMSP, NEM, a decrease in protease activity was observed, which points to the presence of serine and cysteine proteases (data not shown).
Fig. 5. Spectrophotometric characteristics of complexes of actinomycin D with DNA and actinomycin D with a nucleoproteid reconstructed from DNA and acid-soluble proteins of liver mitochondria. (A) liver mitochondrial protein and nDNA. (B) protein and lDNA. 1; free actinomycin D (50 mg/ml). 2; actinomycin D and DNA (70 mg/ml). 3; actinomycin D and nucleoproteid (protein:DNAZ1.5:1).
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Fig. 6. Differential spectra of DNA-actinomycin D and DNA– protein-actinomycin D against actinomycin D. 1, lDNA-actinomycin D; 2, lDNA–protein-actinomycin D (protein:DNAZ1.5:1); 3, lDNA–protein-actinomycin D after treatment with 1 M NaCl (control: actinomycin D and 1 M NaCl).
In order to determine the amount of proteases associated with acid-soluble proteins and their molecular masses, these proteins were subjected to enzyme-electrophoresis in 15% SDS-polyacrylamide gel containing casein. After electrophoresis, the gel was incubated as described in Section 2. After the staining of the gel, proteins and casein were colored, whereas in the sites of protease location white spots were formed because of hydrolysis of acrylamide-bound casein by proteases. In the electrophoregram presented in Fig. 8, seven proteases are seen associated with acid-soluble proteins of liver and spleen mitochondria. The proteases have molecular masses between 20 and 90 kDa. As seen from the Figure, proteases of mitochondrial acid-soluble proteins of spleen substantially differ from proteases of those of liver. In particular, in the acid-soluble proteins from spleen mitochondria a protease of 63 kDa is absent and proteases with molecular masses 33 and 27–29 kDa are more active than the same proteases in the liver mitochondrial proteins.
4. Discussion Recently a high vulnerability of mtDNA has been reported by a number of authors. Compared to the nDNA, the mtDNA is more susceptible not only to ROS that are formed in mitochondria but also to
Fig. 7. Effect of DNA on the activity of proteases of liver mitochondrial acid-soluble proteins. Proteins (50 mg) in 0.04 M Tris–HCl buffer, pH 8.0, were incubated for 1.5 h at 37 8C in the absence and in the presence of denatured or native mtDNA or nDNA. Lane 1, proteins before incubation; Lane 2, proteins incubated in the absence of DNA; Lanes 3 and 4, proteins incubated in the presence of denatured mtDNA and nDNA; Lanes 5 and 6, proteins incubated in the presence of native mtDNA and nDNA.
various exogenous agents. Exogenous and endogenous factors cause in mtDNA substantially more lesions than in a nDNA fragment equal in size to mtDNA (Richter et al., 1988; Richter, 1992; Sawyer and Van Houten, 1999; Marcelino and Thilly, 1999). The frequency of mutations and deletions in mtDNA is 10–25 times higher than in nDNA (Ozawa, 1997; Marcelino and Thilly, 1999). It was suggested that one of the causes of the high sensitivity of mtDNA to damaging influences of ROS and other genotoxic factors is the absence of histones in mitochondria (Richter, 1992; Wallace, 1992; Higuchi and Linn, 1995). Because of this mitochondrial DNA is not protected by proteins and does not form a densely packed nucleoproteid as it is the case in the nuclei.
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Fig. 8. Enzyme-electrophoresis of liver and spleen mitochondrial acid-soluble proteins. After electrophoresis the gel was incubated for 48 h at 37 8C for determination of protease activity. Lane 1, protein markers; Lane 2, protease activity of liver mitochondrial acidsoluble proteins; Lane 3, protease activity of spleen mitochondrial acid-soluble proteins.
However, our present study showed that in liver and spleen mitochondria there are proteins extractable by 0.2 M H2SO4 which is usually used to extract basic nuclear proteins, histones. These acid-soluble proteins were represented in liver and spleen mitochondria by more than 20 polypeptides (Fig. 1). It should be noted that two of them possessed an electrophoretic mobility comparable with that of H1 and H2B histones. Earlier Olszewska and Tait (1980) have isolated basic proteins similar to histones in electrophoretic mobility from the mitochondrial replication complex Paramecium aurelia by extraction with 0.25 M HCl. Pavco and Van Tuyle (1985) obtained from liver mitochondria a histone-like protein P16
binding to mtDNA in the region of the D-loop. Histone-like proteins were also isolated from mitochondria of yeast and Xenopus laevis (Caron et al., 1979; Mignotte et al., 1985). Some later, using chromatography on DEAE-cellulose, Yamada et al. (1991) isolated histone-like protein HMBH possessing an electrophoretic mobility near to that of histone H2B from bovine heart mitochondria. The protein HMBH bears close resemblance to histone-like protein HM from mitochondria of yeast and some less resemblance to HU of E. coli, P16 of liver mitochondria, and mtSSB of Xenopus laevis mitochondria. Yamada et al. (1991) also isolated two histone-like proteins from an acid extract of bovine heart mitochondria using chromatography on Bio-Rex 70 and Bio-Gel P-60. One of these proteins also had an electrophoretic mobility close to that of histone H2B. Possibly, among the proteins we have isolated from liver and spleen mitochondria (Fig. 1), there are histone-like proteins found in the mitochondria of other cells. It is known that in the presence of 0.2 M H2SO4, histones, the proteins that possess basic properties, are extracted from cell nuclei (Bonner et al., 1968). It can be assumed therefore that a great part of the proteins we have extracted from mitochondria are also basic proteins. The acid-soluble mitochondrial proteins we have isolated possess a high DNA affinity and form nucleoproteid complexes at the physiological concentration of NaCl. The DNA–protein complex formation ceases completely on increasing the NaCl concentration up to 0.5 M. It has been known that dissociation of the linker histone H1 from thymus nucleohistones occurs at about 0.4–0.5 M NaCl (Bonner et al., 1968).Thus, acidsoluble proteins of mitochondria behave in the same manner as histones with respect to forming DNA– protein complexes. Possibly, these proteins fulfil in mitochondria the functions of histones. Recently it has been found that in mammalian mitochondria there are RNA-binding proteins which are likely to be involved in the initiation of transcription. It should be noted that two of these proteins are bound to the inner mitochondrial membrane (Koc and Spremulli, 2003). It has been known that in mammalian mitochondria there are DNA-binding proteins involved in mtDNA functioning. These are transcription factor 1 (mtTF1), transcription factor A of mitochondria (TFAM), and polymerase gamma (POLG) (Fisher et al., 1991;
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Garrido et al., 2003). Recently it has been shown that an ATP-dependent protease, Lon protease, an analoque of La protease (product of the Lon gene) from E. coli is present in the mitochondria of various mammalian tissues (Wang et al., 1993). The presence of numerous proteases in mitochondria has been well known (Bota and Davies, 2001, review). Lon protease is capable of binding to mtDNA and seems to participate in the degradation of proteins controlling mtDNA replication and gene expression (Fu and Markovitz, 1998). In our studies we found that, together with acid-soluble proteins of mitochondria, also proteases are isolated that cleave different fractions of these proteins at different intensities. We have shown for the first time that proteases isolated together with mitochondrial acid-soluble proteins are activated by DNA. The functions of these proteases have not yet been studied. It is possible that proteases we have found participate in the control of mtDNA functioning, like Lon protease. Earlier we detected proteases tightly bound to thymus nuclear histones. These proteases are H1 histonespecific and are activated by DNA containing breaks or denatured regions (Kutsyi and Gaziev, 1994). The activity of histone-associated proteases considerably increases in the nuclei of a regenerating rat liver in the period of intensive transcription and replication of DNA as well as in thymus nuclei upon various influences leading to DNA degradation (exposure to g-radiation, hydrocortisone injection) (Kutsyi et al., 1992; Kutsyi and Gaziev, 1994). When histoneassociated proteases cleave histone H1 in the nuclei, they participate in the decompaction of the highly ordered structure of chromatin and thus facilitate the access of enzymes of transcription, replication, repair or degradation to DNA. Possibly, DNA-dependent proteases extracted together with acid-soluble proteins of mitochondria provide access of various enzymes to mtDNA thus being involved in mtDNA replication, transcription, repair, and degradation. The ability of these proteases to bind to DNA and their activation by DNA, especially by a denatured DNA seems to point to the participation of these enzymes in the functioning of mtDNA. At present, it is absolutely unclear what is the role of acid-soluble proteins we have detected and their accompanying proteases including DNA-activated ones in the functioning of mitochondria and
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mtDNA. Because mitochondrial acid-soluble proteins, like histones, are capable of binding to DNA and forming nucleoproteid complexes it may be assumed that these proteins perform in the mitochondria a function similar to that of histones in the nuclei. When binding to mtDNA, acid-soluble proteins probably influence the structural organization and the functional activity of mtDNA. Because proteases that are isolated together with acid-soluble proteins intensively cleave these proteins it can be suggested that DNA, via DNA-activated proteases, can regulate the number of basic proteins and thus control its own structural organization and functional activity. It is also probable that DNA-activated proteases are involved in mtDNA replication and transcription, while cleaving repressor proteins. It is suggested that mitochondria in which DNA is severely damaged undergo elimination. It is quite possible that the DNAactivated proteases we have detected participate in the elimination of mitochondria, the process triggered by damaged mtDNA.
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Muller, W., Crothers, D.M., 1968. Studies of the binding of actinomycin and related compounds to DNA. J. Mol. Biol. 35, 251–290. Olszewska, E., Tait, A., 1980. Mitochondrial chromatin in Paramecium aurelia. Mol. Gen. Genet. 178, 453–457. Ozawa, T., 1997. Genetic and functional changes in mitochondria associated with aging. Physiol. Rev. 77, 425–464. Pavco, P.A., Van Tuyle, G.C., 1985. Purification and general properties of the DNA-binding protein (P16) from rat liver mitochondria. J. Cell Biol. 100, 258–264. Pedersen, P.L., Greenawalt, J.W., Reynafarje, B., Hullihen, J., Decker, G.L., Soper, J.W., Bustamente, E., 1978. Preparation and characterisationof mitochondria and submitochondrial particles of rat liver and liver-derived tissues, in: Prescott, D.M. (Ed.), Methods in Cell Biology, vol. 20. Acadamic Press, New York, pp. 411–481. Potter, D.A., Fostel, J.M., Berninger, M., Pardue, M.L., Cech, T.R., 1980. DNA–protein interactions in the Drosophilla melanogaster mitochondrial genome as deduced from trimethylpsoralen crosslinking patterns. Proc. Natl Acad. Sci. USA 77, 4118–4122. Richter, C., 1992. Reactive oxygen and DNA damage in mitochondria. Mutat. Res. 275, 249–255. Richter, C., Park, J.-W., Ames, B.N., 1988. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl Acad. Sci. USA 85, 6465–6467. Rickwood, D., Chambers, J.A.A., Barat, M., 1981. Isolation and preliminary characterisation of DNA–protein complexes from the mitochondria of Saccharomyces cerevisiae. Exp. Cell Res. 133, 1–13. Sawyer, D.E., Van Houten, B., 1999. Repair of DNA damage in mitochondria. Mutat. Res. 434, 161–176. Suter, M., Richter, C., 1999. Fragmented mitochondrial DNA is the predominant carrier of oxidized DNA bases. Biochemistry 38, 459–464. Takamatsu, C., Umeda, S., Ohsato, T., Ohno, T., Abe, Y., Fukuoh, A., Shinagawa, H., Hamasaki, N., Kang, D., 2002. Regulation of mitochondrial D-loops by transcription factor A and single-stranded DNA-binding protein. Eur. Mol. Biol. Org. Rep. 18, 451–456. Van Tuyle, G.C., Kalf, G.F., 1972. Isolation of membrane-DNA– RNA complex from rat liver mitochondria. Arch. Biochem. Biophys. 149, 425–434. Van Tuyle, G.C., McPherson, M.L., 1979. A compact form of rat liver mitochondrial DNA stabilized by bound proteins. J. Biol. Chem. 254, 6044–6053. Wallace, D.C., 1992. Diseases of the mitochondrial DNA. Ann. Rev. Biochem. 61, 1175–1212. Wang, N., Gottesman, S., Willingham, M.C., Gottesman, M., Maurizi, M., 1993. A human mitochondrial ATP-dependent protease that is highly homologous to bacterial Lon protease. Proc. Natl Acad. Sci. USA 90, 11247–11251. Yamada, E.W., Dotzlaw, H., Huzel, N.J., 1991. Isolation of histonelike proteins from mitochondria of bovine heart. Prepar. Biochem. 21, 11–23.
DNA-binding proteins of mammalian mitochondria Michael P. Kutsyi*, Natalia A. Gouliaeva, Elena A. Kuznetsova, Azhub I. Gaziev Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, 142290, Russia Received 15 June 2004; received in revised form 1 September 2004; accepted 9 September 2004
Abstract Acid-soluble proteins were isolated from liver and spleen mitochondria and their ability to form complexes with DNA was investigated. According to electrophoresis data, acid-soluble proteins include about 20 polypeptides ranging in the molecular mass from 10 to 120 kDa. It was found that acid-soluble proteins form stable DNA–protein complexes at a physiological NaCl concentration. Different polypeptides possess different degrees of DNA affinity. There is no significant difference between DNA-binding proteins of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In the presence of 5 mg of DNA most polypeptides were bound to DNA, and further increase in DNA amount affected little the binding of proteins to DNA. There was no distinct difference in DNA–protein complex formation of liver mitochondrial acid-soluble proteins with nDNA or mtDNA. Also, it was detected that with these mitochondrial acid-soluble proteins, proteases that specifically cleave these proteins are associated. It was shown for the first time that these proteases are activated by DNA. DNA-binding proteins including DNA-activated mitochondrial proteases are likely to participate in the regulation of the structural organization and functional activity of mitochondrial DNA. q 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved. Keywords: Mitochondria; DNA-binding proteins; Acid-soluble proteins; Histones; Proteases
1. Introduction In mammalian somatic cells, the mitochondrial DNA (mtDNA) is represented by numerous copies (from 1 to several thousands) of double-stranded circular molecules of 16.5 kb (Anderson et al., 1981; Bibb et al., 1981; Takamatsu et al., 2002). DNA in mitochondria is mostly associated with the inner membrane (Adams et al., 1980; Potter et al., 1980). The mtDNA interacts with proteins of the inner
* Corresponding author. Fax: C7 967 330553. E-mail address: [email protected] (M.P. Kutsyi).
mitochondrial membrane via particular D-loop regions (Potter et al., 1980). MtDNA compared to nuclear DNA (nDNA) is more vulnerable to reactive oxygen species (ROS) formed in the mitochondrial respiratory chain. The number of oxidized bases in mtDNA is about 16 times as high as in nDNA (Richter et al., 1988; Richter, 1992). Mutations and deletions in mtDNA may be a cause of aging and development of degenerative diseases in the organism (Wallace, 1992; Cortopassi et al., 1992). It has been suggested that a major contributor to the high sensitivity of mtDNA to damaging ROS influences is the absence of histones in mitochondria. Therefore, in intact mitochondria DNA is not covered by histones and other proteins, as it is
1567-7249/$ - see front matter q 2004 Elsevier B.V. and Mitochondria Research Society. All rights reserved. doi:10.1016/j.mito.2004.09.002
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the case in the nuclei (Richter, 1992; Wallace, 1992; Higuchi and Linn, 1995). At the same time, some authors have shown the presence of mtDNA–protein complexes, in which protein is tightly associated with DNA and this linkage is stable to the action of ionic detergents and high concentrations of NaCl. In particular, DNA–protein complexes have been isolated from rat liver mitochondria after their lysis with sarkosyl (Van Tuyle and Kalf, 1972) or with BRIJ 58 in the presence of sodium deoxycholate and 0.5 M NaCl (Van Tuyle and McPherson, 1979). According to electron microscopy data, mtDNA molecules are organized as rosette-like structures with a dense central core (Van Tuyle and McPherson, 1979). They are maintained in the highly folded state by associated proteins. DNA–protein complexes have also been isolated from the yeast S. cerevisiae mitochondria (Rickwood et al., 1981; Miyakawa et al., 1987). From liver mitochondria, a histone-like protein of 16 kDa molecular mass has been isolated which interacts with nucleotide sequences in the region of the D-loop of mtDNA (Pavco and Van Tuyle, 1985). Histone-like proteins have also been obtained from the mitochondria of heart (Yamada et al., 1991), yeast (Caron et al., 1979), and Xenopus laevis (Mignotte et al., 1985). Recently it has been shown that multiple mtDNA molecules are organized into discrete DNA–protein complexes called ‘nucleoids’ (Miyakawa et al., 1987; Garrido et al., 2003). At the present time, the protein composition of mitochonrial nucleoids and the mechanisms by which mtDNA functions within this dynamic DNA–protein structure have not yet been completely elucidated. It has been shown that nucleoids from the mitochondria of primary human fibroblast cells, 143B osteosarcoma and A549 adenocarcinoma cells contain mitochondrial gamma-polymerase, the transcriptional factor A of mitochondria, and mitochondrial single-stranded DNA binding proteins (Garrido et al., 2003). Probably, proteins loosely bound to DNA also participate in the packing of mtDNA within the nucleoid. When DNA or DNA–protein complexes are isolated under rigorous conditions with the use of ionic detergents or high salt concentrations, the proteins loosely bound to DNA dissociate and hence are not detected in isolated preparations of DNA and DNA–protein complexes. Recently, proteins binding to RNA have been shown
to be present in mammalian mitochondria (Koc and Spremulli, 2003). In the present work, a spectrum of acid-soluble DNA-binding proteins of rat liver and spleen mitochondria is presented and their ability to form complexes with DNA is shown. Also it is shown that these acid-soluble proteins contain proteases including DNA-activated ones.
2. Materials and methods 2.1. Materials All chemical reagents were of analytical grade. All reagents for polyacrylamide gel electrophoresis (PAGE), calf thymus DNA, DNaase, RNaase A, antipain, leupeptin, phenylmethylsulphonyl fluoride (PMSF), N-ethymaleimide (NEM), and digitonin were obtained from Sigma Chemical Company. Proteinase K and sodium dodecyl sulfate (SDS) were obtained from AppliChem GmbH (Germany), lambda DNA from phage isolated from the E. coli W3110 (c/1857 Sam7) strain, protein molecular mass standards (b-galactosidase, 116.0 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 45.0 kDa; lactate dehydrogenase, 35.0 kDa; restriction endonuclease Bsp981, 25.0 kDa; b-lactoglobulin, 18.4 kDa; lysozyme, 14.4 kDa) were obtained from Fermentas MBI. 2.2. Isolation of nuclei and mitochondria Male Wistar rats weighing 180–200 g were used. Rat liver and spleen mitochondria were isolated by differential centrifugation, as described by Pedersen et al. (1978). Mitochondria were purified from lysosomal impurities by treatment with digitonin (Loewenstein et al., 1970). Nuclei from liver and spleen were isolated as described by Blobel and Potter (1966). DNA from liver mitochondria was isolated according to Suter and Richter (1999). 2.3. Extraction of acid-soluble proteins from nuclei and mitochondria Acid-soluble proteins (histones) were extracted from liver and spleen nuclei with 0.2 M H2SO4, as described by Bonner et al. (1968). Histones thus
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obtained were precipitated with ethanol and extracted once again with 0.2 M H2SO4. Analogously, acidsoluble proteins were extracted from liver and spleen mitochondria. Protein concentration was determined according to Lowry et al. (1951). 2.4. Electrophoresis of proteins in polyacrylamide gel Electrophoresis of acid-soluble proteins was performed in 15% SDS-polyacrylamide gel by the method of Laemmly (1970). Enzyme-electrophoresis of proteases associated with acid-soluble proteins of mitochondria was carried out in 15% SDS-polyacrylamide gel containing covalently bound casein according to Kelleher and Juliano (1984) with minor modification. Acid-soluble mitochondrial proteins were dissolved in a buffer containing SDS but with no 2-mercaptoethanol. Samples were not boiled before being applied to gel. After electrophoresis, the gel was washed for 1.5 h with 0.02 M Tris–HCl buffer, pH 8.0 containing 0.5% Triton X-100 to remove SDS. In order to detect protease activity, the gel was incubated for 48 h at 37 8C in 0.02 M Tris– HCl buffer, pH 8.0, containing 25 mM NaCl. After incubation, the gel was stained with Coomassie brilliant blue (CBB R-250). 2.5. Formation of DNA–protein complexes To obtain DNA–protein complexes, different amounts of DNA (2, 5, and 10 mg) were added to 50 mg of acid-soluble mitochondrial proteins in 0.02 M Tris–HCl, pH 7.4, containing 0.15 M NaCl. In order to study the effect of NaCl on the formation of DNA–protein complexes, 0.15–0.5 M NaCl was added to 50 mg of protein and 10 mg of DNA. Thirty minutes later, DNA–protein complexes were sedimented by centrifugation (5000!g, 30 min at 4 8C). Then the pellet and supernatant were subjected to electrophoresis in 15% SDS-polyacrylamide gel. The formation of DNA–protein complexes was also judged from comparing the binding of actinomycin D to free DNA and DNA complexed with protein (Muller and Crothers, 1968). In the case of DNA–protein complex formation, the binding of actinomycin D must be lower than in the case of free DNA.
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2.6. Assay of protease activity In order to determine the activity of proteases associated with acid-soluble mitochondrial proteins, 50 mg of proteins in 0.04 M Tris–HCl buffer, pH 8.0, were incubated for 1.5–3.0 h at 37 8C in the presence or in the absence of 10 mg of native or denatured DNA. DNA was denatured by heating for 10 min at 100 8C and then rapidly cooled. After incubation, samples were subjected to electrophoresis in 15% SDS-polyacrylamide gel. After staining with Coomassie brilliant blue, gels were scanned with subsequent computation. Protease activity was judged from the amount of polypeptides degraded in the course of incubation. In estimating the pH dependence of the activity of proteases associated with mitochondrial acid-soluble proteins, these proteins were incubated for 3 h at 37 8C and the amount of degraded proteins was determined by the method of Lowry et al. (1951).
3. Results 3.1. DNA-binding acid-soluble proteins of mitochondria Acid-soluble proteins were isolated from mitochondria and nuclei of rat liver and spleen by extraction with 0.2 M H2SO4. It should be noted that extraction with 0.2 M H2SO4 is commonly used to isolate basic proteins, histones, from the nuclei (Bonner et al., 1968). As is seen from electrophoregrams in Fig. 1 (lanes 3, 5), the basic proteins from liver and spleen nuclei are represented by five histone fractions, which is consistent with the literature data (Bonner et al., 1968; Hnilica, 1975; Olszewska and Tait, 1980). However, in our experiments the treatment of liver and spleen mitochondria with 0.2 M H2SO4 also gave acid-soluble proteins. Upon electrophoresis in 15% SDS-polyacrylamide gel, these proteins were divided into more than 20 polypeptides with molecular masses of 10–120 kDa. The electrophoretical spectra of acid-soluble proteins from spleen mitochondria are similar to those from liver mitochondria, although there are distinct differences in the ratios between particular peptides (Fig. 1, lanes 2, 4). As upon the treatment of nuclei with sulfuric acid the basic
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Fig. 1. Acid-soluble proteins of mitochondria and nuclei from rat liver and spleen. Lane 1, protein markers; Lane 2, liver mitochondrial proteins; Lane 3, liver nuclear proteins (histones); Lane 4, spleen mitochondrial proteins; Lane 5, spleen nuclear proteins (histones).
proteins, histones, are extracted, it can be assumed that the acid-soluble proteins extracted by the same procedure from mitochondria may mostly possess the properties of basic proteins. 3.2. Formation of nucleoproteid complexes: acid-soluble proteins–DNA In further experiments, it was found that acidsoluble proteins from liver and spleen mitochondria form nucleoproteid complexes with DNA at the physiological NaCl concentration. These complexes are easily isolated by centrifugation from the DNA and proteins that are unable to form complexes and remain in the supernatant (Fig. 2). There is no significant difference between DNA-binding proteins
Fig. 2. Formation of mitochondrial acid-soluble proteins–DNA complexes. MtDNA (10 mg) or nDNA (10 mg) were added to 50 mg of spleen mitochondrial proteins in 0.02 M Tris–HCl buffer, pH 7.4, containing 0.15 M NaCl. After centrifugation, the proteins of pellet (P) and those of supernatant (S) were subjected to electrophoresis. Lane 1; initial proteins (50 mg). Lanes 2 and 3; protein and mtDNA. Lanes 4 and 5; protein and nDNA.
of mitochondria from liver and those from spleen as to their ability to form complexes with mtDNA and nDNA. In studying the dependence of DNA–protein complex formation on the amount of nDNA or mtDNA, it was found that addition of 2 mg of DNA to the reaction mixture resulted in the binding of substantial amounts of mitochondrial acid-soluble proteins (Fig. 3). In the presence of 5 mg of DNA most polypeptides were bound to DNA, and further increase of DNA amount to 10 mg influenced little the binding of proteins to DNA. There was no distinct difference in DNA–protein complex formation of liver mitochondrial acid soluble proteins with nDNA or mtDNA (Fig. 3). The study of the NaCl concentration dependence of the binding of liver
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Fig. 3. Dependence of acid-soluble protein–DNA complex formation on DNA concentration. MtDNA or nDNA (2, 5, 10 mg) were added to 50 mg of liver mitochondrial proteins in 0.02 M Tris–HCl buffer, pH 7.4, containing 0.15 M NaCl. After centrifugation, the proteins of pellet (P) and those of supernatant (S) were subjected to electrophoresis. (A) mtDNA–protein complexes; (B) nDNA–protein complexes. Lane 1; initial proteins (50 mg). Lanes 2 and 3; proteins and 2 mg DNA. Lanes 4 and 5; proteins and 5 mg DNA. Lanes 6 and 7; proteins and 10 mg DNA.
mitochondrial acid-soluble proteins to DNA showed that when NaCl concentration increases, the formation of nucleoproteid complexes gradually decreases to stop completely at 0.5 M NaCl (Fig. 4). The formation of DNA–protein complexes was also determined from the capacity of proteins to compete with actinomycin D for DNA-binding sites. The binding of actinomycin D to DNA was known to be accompanied by a change in its optical properties (Muller and Crothers, 1968). In the formation of a DNA–protein complex, the binding of actinomycin D must be lower for the DNA–protein complex than for free DNA. The absorption spectra of free actinomycin D, the nDNA-actinomycin D complex and a reconstructed nucleoproteid–actinomycin D complex are presented in Fig. 5A. It appeared that addition of acidsoluble proteins from liver mitochondria to nDNA at the ratio 1.5:1 reduced actinomycin binding by about 40%, which points to the formation of a DNA–protein
complex. When lDNA was used, a nearly the same spectrophotometric characteristics of the DNA–protein complex was observed (Fig. 5B). Lambda DNA from the phage isolated from the E. coli W3110 (c/1857 Sam7) strain consists of 48502 bp, it is nearly 3 times as long as mitochondrial DNA. The study of the differential spectra of DNA-actinomycin D and DNA–protein-actinomycin D against actinomycin D (Fig. 6) showed changes in the character of the spectral curve for the complex DNA–protein-actinomycin D as compared to the DNA-actinomycin D complex. These differences are due to different amounts of actinomycin D bound to free DNA and the DNA–protein complex. The differential spectra presented show distinctly that to DNA complexed with protein a substantially less amount of actinomycin D is bound than to free DNA since the binding site on DNA in the DNA–protein complex are partially occupied by protein. Addition of NaCl to
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a concentration of 1 M resulted in dissociation of the DNA–protein complex, which is indicated by a change in the spectral curve (it tends to became a direct line). Thus, the results point to that acid-soluble mitochondrial proteins form complexes with DNA. 3.3. Proteases associated with acid-soluble proteins of mitochondria. DNA-activated proteases
Fig. 4. Dependence of acid-soluble protein–DNA complex formation on NaCl concentration. NaCl (0.15 M, 0.35 M, 0.42 M, 0.5 M) and 10 mg nDNA were added to 50 mg of liver mitochondrial proteins in 0.02 M Tris–HCl buffer, pH 7.4. After centrifugation, proteins of the pellet (P) and those of the supernatant (S) were subjected to electrophoresis. Lane 1; initial proteins (50 mg). Lanes 2 and 3; proteins and nDNA with 0.15 M NaCl. Lanes 4 and 5; proteins and nDNA with 0.35 M NaCl. Lanes 6 and 7; proteins and nDNA with 0.42 M NaCl. Lanes 8 and 9; proteins and nDNA with 0.5 M NaCl.
It has been well known that mitochondria contain many proteases involved in the metabolism of mitochondrial proteins (Bota and Davies, 2001, review). In our investigation it was revealed that along with acid-soluble proteins, also proteases were extracted that cleaved various polypeptides of these proteins at various intensity (Fig. 7). We showed for the first time that the activity of these proteases increased in the presence of DNA and that denatured nDNA and mtDNA activated them to a higher degree than native nDNA and mtDNA do (Fig. 7). The intensity of cleavage of the acid-soluble proteins by these proteases was higher than the cleavage of other mitochondrial proteins. The optimum activity of proteases associated with acid-soluble proteins was observed at pH 8.0 (data not shown). When acid-soluble proteins from liver mitochondria were incubated with the protease inhibitors antipain, leupeptin, PMSP, NEM, a decrease in protease activity was observed, which points to the presence of serine and cysteine proteases (data not shown).
Fig. 5. Spectrophotometric characteristics of complexes of actinomycin D with DNA and actinomycin D with a nucleoproteid reconstructed from DNA and acid-soluble proteins of liver mitochondria. (A) liver mitochondrial protein and nDNA. (B) protein and lDNA. 1; free actinomycin D (50 mg/ml). 2; actinomycin D and DNA (70 mg/ml). 3; actinomycin D and nucleoproteid (protein:DNAZ1.5:1).
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Fig. 6. Differential spectra of DNA-actinomycin D and DNA– protein-actinomycin D against actinomycin D. 1, lDNA-actinomycin D; 2, lDNA–protein-actinomycin D (protein:DNAZ1.5:1); 3, lDNA–protein-actinomycin D after treatment with 1 M NaCl (control: actinomycin D and 1 M NaCl).
In order to determine the amount of proteases associated with acid-soluble proteins and their molecular masses, these proteins were subjected to enzyme-electrophoresis in 15% SDS-polyacrylamide gel containing casein. After electrophoresis, the gel was incubated as described in Section 2. After the staining of the gel, proteins and casein were colored, whereas in the sites of protease location white spots were formed because of hydrolysis of acrylamide-bound casein by proteases. In the electrophoregram presented in Fig. 8, seven proteases are seen associated with acid-soluble proteins of liver and spleen mitochondria. The proteases have molecular masses between 20 and 90 kDa. As seen from the Figure, proteases of mitochondrial acid-soluble proteins of spleen substantially differ from proteases of those of liver. In particular, in the acid-soluble proteins from spleen mitochondria a protease of 63 kDa is absent and proteases with molecular masses 33 and 27–29 kDa are more active than the same proteases in the liver mitochondrial proteins.
4. Discussion Recently a high vulnerability of mtDNA has been reported by a number of authors. Compared to the nDNA, the mtDNA is more susceptible not only to ROS that are formed in mitochondria but also to
Fig. 7. Effect of DNA on the activity of proteases of liver mitochondrial acid-soluble proteins. Proteins (50 mg) in 0.04 M Tris–HCl buffer, pH 8.0, were incubated for 1.5 h at 37 8C in the absence and in the presence of denatured or native mtDNA or nDNA. Lane 1, proteins before incubation; Lane 2, proteins incubated in the absence of DNA; Lanes 3 and 4, proteins incubated in the presence of denatured mtDNA and nDNA; Lanes 5 and 6, proteins incubated in the presence of native mtDNA and nDNA.
various exogenous agents. Exogenous and endogenous factors cause in mtDNA substantially more lesions than in a nDNA fragment equal in size to mtDNA (Richter et al., 1988; Richter, 1992; Sawyer and Van Houten, 1999; Marcelino and Thilly, 1999). The frequency of mutations and deletions in mtDNA is 10–25 times higher than in nDNA (Ozawa, 1997; Marcelino and Thilly, 1999). It was suggested that one of the causes of the high sensitivity of mtDNA to damaging influences of ROS and other genotoxic factors is the absence of histones in mitochondria (Richter, 1992; Wallace, 1992; Higuchi and Linn, 1995). Because of this mitochondrial DNA is not protected by proteins and does not form a densely packed nucleoproteid as it is the case in the nuclei.
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Fig. 8. Enzyme-electrophoresis of liver and spleen mitochondrial acid-soluble proteins. After electrophoresis the gel was incubated for 48 h at 37 8C for determination of protease activity. Lane 1, protein markers; Lane 2, protease activity of liver mitochondrial acidsoluble proteins; Lane 3, protease activity of spleen mitochondrial acid-soluble proteins.
However, our present study showed that in liver and spleen mitochondria there are proteins extractable by 0.2 M H2SO4 which is usually used to extract basic nuclear proteins, histones. These acid-soluble proteins were represented in liver and spleen mitochondria by more than 20 polypeptides (Fig. 1). It should be noted that two of them possessed an electrophoretic mobility comparable with that of H1 and H2B histones. Earlier Olszewska and Tait (1980) have isolated basic proteins similar to histones in electrophoretic mobility from the mitochondrial replication complex Paramecium aurelia by extraction with 0.25 M HCl. Pavco and Van Tuyle (1985) obtained from liver mitochondria a histone-like protein P16
binding to mtDNA in the region of the D-loop. Histone-like proteins were also isolated from mitochondria of yeast and Xenopus laevis (Caron et al., 1979; Mignotte et al., 1985). Some later, using chromatography on DEAE-cellulose, Yamada et al. (1991) isolated histone-like protein HMBH possessing an electrophoretic mobility near to that of histone H2B from bovine heart mitochondria. The protein HMBH bears close resemblance to histone-like protein HM from mitochondria of yeast and some less resemblance to HU of E. coli, P16 of liver mitochondria, and mtSSB of Xenopus laevis mitochondria. Yamada et al. (1991) also isolated two histone-like proteins from an acid extract of bovine heart mitochondria using chromatography on Bio-Rex 70 and Bio-Gel P-60. One of these proteins also had an electrophoretic mobility close to that of histone H2B. Possibly, among the proteins we have isolated from liver and spleen mitochondria (Fig. 1), there are histone-like proteins found in the mitochondria of other cells. It is known that in the presence of 0.2 M H2SO4, histones, the proteins that possess basic properties, are extracted from cell nuclei (Bonner et al., 1968). It can be assumed therefore that a great part of the proteins we have extracted from mitochondria are also basic proteins. The acid-soluble mitochondrial proteins we have isolated possess a high DNA affinity and form nucleoproteid complexes at the physiological concentration of NaCl. The DNA–protein complex formation ceases completely on increasing the NaCl concentration up to 0.5 M. It has been known that dissociation of the linker histone H1 from thymus nucleohistones occurs at about 0.4–0.5 M NaCl (Bonner et al., 1968).Thus, acidsoluble proteins of mitochondria behave in the same manner as histones with respect to forming DNA– protein complexes. Possibly, these proteins fulfil in mitochondria the functions of histones. Recently it has been found that in mammalian mitochondria there are RNA-binding proteins which are likely to be involved in the initiation of transcription. It should be noted that two of these proteins are bound to the inner mitochondrial membrane (Koc and Spremulli, 2003). It has been known that in mammalian mitochondria there are DNA-binding proteins involved in mtDNA functioning. These are transcription factor 1 (mtTF1), transcription factor A of mitochondria (TFAM), and polymerase gamma (POLG) (Fisher et al., 1991;
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Garrido et al., 2003). Recently it has been shown that an ATP-dependent protease, Lon protease, an analoque of La protease (product of the Lon gene) from E. coli is present in the mitochondria of various mammalian tissues (Wang et al., 1993). The presence of numerous proteases in mitochondria has been well known (Bota and Davies, 2001, review). Lon protease is capable of binding to mtDNA and seems to participate in the degradation of proteins controlling mtDNA replication and gene expression (Fu and Markovitz, 1998). In our studies we found that, together with acid-soluble proteins of mitochondria, also proteases are isolated that cleave different fractions of these proteins at different intensities. We have shown for the first time that proteases isolated together with mitochondrial acid-soluble proteins are activated by DNA. The functions of these proteases have not yet been studied. It is possible that proteases we have found participate in the control of mtDNA functioning, like Lon protease. Earlier we detected proteases tightly bound to thymus nuclear histones. These proteases are H1 histonespecific and are activated by DNA containing breaks or denatured regions (Kutsyi and Gaziev, 1994). The activity of histone-associated proteases considerably increases in the nuclei of a regenerating rat liver in the period of intensive transcription and replication of DNA as well as in thymus nuclei upon various influences leading to DNA degradation (exposure to g-radiation, hydrocortisone injection) (Kutsyi et al., 1992; Kutsyi and Gaziev, 1994). When histoneassociated proteases cleave histone H1 in the nuclei, they participate in the decompaction of the highly ordered structure of chromatin and thus facilitate the access of enzymes of transcription, replication, repair or degradation to DNA. Possibly, DNA-dependent proteases extracted together with acid-soluble proteins of mitochondria provide access of various enzymes to mtDNA thus being involved in mtDNA replication, transcription, repair, and degradation. The ability of these proteases to bind to DNA and their activation by DNA, especially by a denatured DNA seems to point to the participation of these enzymes in the functioning of mtDNA. At present, it is absolutely unclear what is the role of acid-soluble proteins we have detected and their accompanying proteases including DNA-activated ones in the functioning of mitochondria and
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mtDNA. Because mitochondrial acid-soluble proteins, like histones, are capable of binding to DNA and forming nucleoproteid complexes it may be assumed that these proteins perform in the mitochondria a function similar to that of histones in the nuclei. When binding to mtDNA, acid-soluble proteins probably influence the structural organization and the functional activity of mtDNA. Because proteases that are isolated together with acid-soluble proteins intensively cleave these proteins it can be suggested that DNA, via DNA-activated proteases, can regulate the number of basic proteins and thus control its own structural organization and functional activity. It is also probable that DNA-activated proteases are involved in mtDNA replication and transcription, while cleaving repressor proteins. It is suggested that mitochondria in which DNA is severely damaged undergo elimination. It is quite possible that the DNAactivated proteases we have detected participate in the elimination of mitochondria, the process triggered by damaged mtDNA.
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