Inhibition of Ribonuclease P Activity by Retinoids
1998; Elsevier BV; Volume: 273; Issue: 38 Linguagem: Inglês
10.1074/jbc.273.38.24375
ISSN1083-351X
AutoresEvangelia Papadimou, Sophia Georgiou, D. Tsambaos, Denis Drainas,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoThe effect of two naturally occurring (retinol and all-trans retinoic acid) and two synthetic (isotretinoin and acitretin) analogs of vitamin A (retinoids) on tRNA biogenesis was investigated employing the RNase P of Dictyostelium discoideum as an in vitroexperimental system. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5′ end. All retinoids tested revealed a dose-dependent inhibition of RNase P activity, indicating that these compounds may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classical competitive inhibitors. The K i values determined were 1475 μmfor retinol, 15 μm for all-trans retinoic acid, 20 μm for isotretinoin, and 8.0 μmfor acitretin. On the basis of these values acitretin is a 184, 2.5, and 1.9 times more potent inhibitor, as compared with retinol, isotretinoin, and all-trans retinoic acid, respectively. Taking into account that retinoids share no structural similarities to precursor tRNA, it is suggested that their kinetic behavior reflects allosteric interactions of these compounds with hydrophobic site(s) of D. discoideum RNase P. The effect of two naturally occurring (retinol and all-trans retinoic acid) and two synthetic (isotretinoin and acitretin) analogs of vitamin A (retinoids) on tRNA biogenesis was investigated employing the RNase P of Dictyostelium discoideum as an in vitroexperimental system. RNase P is an ubiquitous and essential enzyme that endonucleolytically cleaves all tRNA precursors to produce the mature 5′ end. All retinoids tested revealed a dose-dependent inhibition of RNase P activity, indicating that these compounds may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that all retinoids behave as classical competitive inhibitors. The K i values determined were 1475 μmfor retinol, 15 μm for all-trans retinoic acid, 20 μm for isotretinoin, and 8.0 μmfor acitretin. On the basis of these values acitretin is a 184, 2.5, and 1.9 times more potent inhibitor, as compared with retinol, isotretinoin, and all-trans retinoic acid, respectively. Taking into account that retinoids share no structural similarities to precursor tRNA, it is suggested that their kinetic behavior reflects allosteric interactions of these compounds with hydrophobic site(s) of D. discoideum RNase P. Retinoids, a group of natural and synthetic analogs of vitamin A, play an essential role in vision, growth, and reproduction as well as exhibiting striking effects on cell proliferation, differentiation, and pattern formation during development (1Tsambaos D. Zimmerman B. Roenigk H.H. Maibach H.I. Effects of Synthetic Retinoids on Cellular Systems in Psoriasis. Marcel Dekker Inc., New York1991: 659-707Google Scholar, 2Ross A.C. FASEB J. 1993; 7: 317-327Crossref PubMed Scopus (170) Google Scholar). The discovery that members of the steroid/thyroid hormone receptor superfamily are nuclear retinoic acid-binding proteins tremendously improves our understanding of the mechanisms that mediate the regulatory action of retinoids on gene expression (3DeLuca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (812) Google Scholar, 4van der Saag P.T. Eur. J. Clin. Nutr. 1996; 50: 24-28PubMed Google Scholar, 5Botling J. Castro D.S. Oberg F. Nilsson K. Perlmann T. J. Biol. Chem. 1997; 272: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 6Leid M. Kastner P. Chambon P. Trends Biochem Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar). The retinoid receptors are ligand-activated, DNA-binding, trans-acting, transcription factors (7Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2841) Google Scholar, 8Minucci S. Leid M. Toyama R. Saint-Jeannet J.P. Peterson V.J. Horn V. Ishmael J.E. Bhattacharyya N. Dey A. Dawid I.B. Ozato K. Mol. Cell. Biol. 1997; 17: 644-655Crossref PubMed Scopus (148) Google Scholar).Due to their ability to regulate cell differentiation and suppress or reverse the malignant phenotype, retinoids have a potential use as chemopreventive and chemotherapeutic agents in cancers of skin and other organs (9Lippman, S. M., Heyman, R. A., Kurie, J. M., Benner, S. E., and Hong, W. K. (1995) J. Cell. Biochem.22, (Suppl.) 1–10Google Scholar, 10Muindi J.R. Cancer Treat. Res. 1996; 870: 305-342Crossref Scopus (11) Google Scholar, 11Sankaranarayanan R. Mathew B. IARC Sci. Publ. 1996; 139: 47-59PubMed Google Scholar). Moreover, oral synthetic retinoids are presently successfully applied in the management of severe and recalcitrant dermatoses, which were previously regarded as frustrating therapeutic problems (12Wieder J.M. Lowe N.J. Dermatol. Clin. 1995; 13: 891-896Abstract Full Text PDF PubMed Google Scholar, 13Lacour M. Mehta-Nikhar B. Atherton D.J. Harper J.I. Br. J. Dermatol. 1996; 134: 1023-1029Crossref PubMed Scopus (121) Google Scholar, 14Plewig G. Albrecht G. Henz B.M. Meigel W. Schöpf E. Stadler R. Hautarzt. 1997; 48: 881-885Crossref PubMed Scopus (11) Google Scholar).RNase P is a key enzyme in tRNA biogenesis, which cleaves all tRNA precursors endonucleolytically to produce the mature 5′ end. RNase P enzymes are composed of both RNA and protein (15Darr S.C. Brown J.W. Pace N.R. Trends Biochem. Sci. 1992; 17: 178-182Abstract Full Text PDF PubMed Scopus (94) Google Scholar). In vitro, RNA subunits of bacterial enzymes are catalytically active in the absence of protein (16Guerrier-Takada C. Gardiner T. Marsh K. Pace N. Altman S. Cell. 1983; 35: 849-857Abstract Full Text PDF PubMed Scopus (2007) Google Scholar) and are the only known RNA catalysts naturally devoted to act in trans (17Symons R.H. Curr. Opin. Struct. Biol. 1994; 4: 322-330Crossref Scopus (54) Google Scholar). Catalytic activity of RNA subunits, in the absence of protein subunits, has not been demonstrated so far for archaea and eukaryotes RNase P. However, the catalytic center of these RNase P enzymes most likely is associated with the RNA subunits. Eukaryotic RNase P activity has been detected in nuclei, mitochondria, and chloroplasts (15Darr S.C. Brown J.W. Pace N.R. Trends Biochem. Sci. 1992; 17: 178-182Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 18Drainas D. Mol. Biol. Rep. 1996; 22: 135-138Crossref Scopus (7) Google Scholar). Recently, the partial purification and characterization of RNase P from the slime mold Dictyostelium discoideum has been reported (18Drainas D. Mol. Biol. Rep. 1996; 22: 135-138Crossref Scopus (7) Google Scholar,19Stathopoulos C. Kalpaxis D.L. Drainas D. Eur. J. Biochem. 1995; 228: 976-980Crossref PubMed Scopus (31) Google Scholar).In the present study we have examined the effect of natural and synthetic retinoids on RNase P using a cell free system from the slime mold D. discoideum as an in vitro model. D. discoideum RNase P is a nuclear enzyme that has low buoyant density in Cs2SO4 gradients possibly due to the occurrence of an unidentified component such as a fatty acid or lipid (19Stathopoulos C. Kalpaxis D.L. Drainas D. Eur. J. Biochem. 1995; 228: 976-980Crossref PubMed Scopus (31) Google Scholar). This hypothesis is consistent with the existence of hydrophobic site(s) on D. discoideum RNase P and perhaps justifies the role of retinoids as potential inhibitors, being hydrophobic molecules acting in the nucleus.RESULTSThe present data show that all retinoids investigated here (Fig. 1) are capable of inhibiting the RNase P activity of D. discoideum. The substrate for RNase P assays was an in vitro labeled transcript of the S. pombe tRNASer gene supSI (20Drainas D. Zimmerly S. Willis I. Söll D. FEBS Lett. 1989; 251: 84-88Crossref PubMed Scopus (26) Google Scholar).The concentration of all-trans retinoic acid (IC50) at which the product formation is reduced by 50% is equal to 80 μm (Fig. 2,A and B). Although not as effective as all-trans retinoic acid, retinol also inhibited RNase P activity; the IC50 was equal to 500 μm (Fig. 2, C and D). The dose response curves for the synthetic retinoids isotretinoin and acitretin were similar to those observed with the natural retinoids (Fig. 3). The IC50 values were 60 μm (Fig. 3, A and B) and 40 μm (Fig. 3, C and D), respectively. It is obvious from these results that acitretin is the strongest inhibitor among the retinoids tested. It is important to note that RNase P purified through the second DE 52 column was inhibited by retinoids to the same extent as RNase P purified through the S-300 column.Figure 3Dose response effect of isotretinoin (Aand B) and acitretin (C and D). A and C, autoradiograms of the cleavage of the supS1 precursor by RNase P in the presence of isotretinoin (A) and acitretin (C).A, lane 1, supS1 alone; lane 2, control; lanes 3–8, incubated RNase P in the presence of 0.01, 0.05, 0.1, 0.2, 0.4, and 0.5 mmisotretinoin, respectively. C, lane 1, supS1 alone; lane 2, control; lanes 3–8, incubated RNase P in the presence of 0.001, 0.01, 0.02, 0.04, 0.08, and 0.1 mm acitretin, respectively.B and D, RNase P activity as a function of the logarithm of increasing concentrations of isotretinoin (B) and acitretin (D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The type of inhibition of D. discoideum RNase P activity by the natural and synthetic retinoids was further elucidated by detailed kinetic analysis. Because the yields of RNase P after gel filtration chromatography are low, for the kinetic analysis we used RNase P fractions obtained from DEAE-cellulose chromatography. Due to the high hydrophobicity of retinoids, all assays were carried out in the presence of 10% Me2SO. At this concentration, Me2SO marginally affects (K i = 4m) the catalytic parameters of D. discoideumRNase P. The initial velocity in the presence or absence of retinoids was determined from the initial slopes of time plots (not shown). Fig. 4 shows double reciprocal plots with increasing concentrations of acitretin. The lowest line in Fig. 4represents the data obtained in the absence of inhibitor and Me2SO (control). The calculated apparentK m (K m,app) and the apparent V max (V max,app) values from this plot are 240 nm and 3 pmol/min, respectively. These values are in agreement with previous values reported from our laboratory (19Stathopoulos C. Kalpaxis D.L. Drainas D. Eur. J. Biochem. 1995; 228: 976-980Crossref PubMed Scopus (31) Google Scholar). In the presence of 10% Me2SO the K m,app and theV max,app values are 250 nm and 3 pmol/min, respectively (Fig. 4). The slopes of the lines in Fig. 4 were replotted against the concentration of acitretin, and the results are shown in the top panel of Fig. 4. The linearity of this replot is indicative of simple competitive inhibition and leads to the graphical determination of K i = 8.0 μm from the negative intercept of the line with the I-axis. Further evidence for simple competitive kinetics comes from the Dixon plot, which is shown in Fig. 5. When the slopes of the lines of Fig. 4 are replotted against 1/[pre-tRNA] (Fig. 5, top panel) they give a straight line passing through the origin. This is further evidence of simple competitive inhibition (21Segel I.H. Enzyme kinetics. John Wiley & Sons, Inc., New York1975: 110Google Scholar). Hill plots were obtained by calculating the log[v′/(v 0-v′)] values at each concentration of pre-tRNA and plotting them as a function of logarithmic retinoid concentration. The molecular interaction coefficient (n) for retinoids was given by the slope of these plots. Fig. 6 shows the Hill plot for acitretin obtained at 100 nm pre-tRNA. Then value calculated from this plot is about equal to 1. These results support the notion that the inhibition of D. discoideum RNase P activity by retinoids involves only one retinoid molecule.Figure 4Double reciprocal plot (1/v versus 1/[pre-tRNA]) for RNase P reaction in the presence of acitretin. The reaction was carried out at each one of the indicated concentrations in the presence or in the absence of inhibitor. All reactions were carried out at 37 °C in 20 μl of buffer D in the presence of 10% Me2SO. •, without inhibitor minus Me2SO; ○, without inhibitor plus Me2SO; ▪, with acitretin at 15 μm; ▵, with acitretin at 25 μm; ▴, with acitretin at 30 μm; ▪, with acitretin at 50 μm; ♦, with acitretin at 75 μm. Top panel, replot of the slopes of the double reciprocal lines versus inhibitor (I) concentrations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Dixon plot for the RNase P reaction in the presence of acitretin. The concentrations of pre-tRNA were as follows: •, 50 nm; ▴, 100 nm; ▵, 125 nm; ■, 150 nm; ▪, 250 nm; ♦, 500 nm. Top panel, replot of the slopes of the Dixon lines versus 1/[pre-tRNA].View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Hill plot of inhibition of the RNase P reaction by acitretin. The molecular interaction coefficient (n) of acitretin was obtained from the slope of this plot. The v′ (in the presence of acitretin) and v 0 (in the absence of acitretin) values were calculated from the corresponding time plots.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The same kinetic examination was carried out for isotretinoin, all-trans retinoic acid, and retinol. All these compounds showed simple competitive inhibition with molecular interaction coefficient equal to 1. The corresponding K i values are given in Table I.Table IEquilibrium constants derived from primary and secondary kinetic plotsCompoundK iμmRetinol1475.0All-trans retinoic acid15.0Isotretinoin20.0Acitretin8.0The K i values are calculated from the negative intercept of the slope replots. Open table in a new tab To address the question of whether retinoids may be capable of affecting also other nucleases, we investigated the effect of retinol, all-trans retinoic acid, isotretinoin, and acitretin on RNase A from bovine pancreas and found that these compounds exert no effect on RNase A activity (data not shown).DISCUSSIONThe study of the action of retinoids is of essential importance because these compounds are involved in gene expression, cell growth, and differentiation (1Tsambaos D. Zimmerman B. Roenigk H.H. Maibach H.I. Effects of Synthetic Retinoids on Cellular Systems in Psoriasis. Marcel Dekker Inc., New York1991: 659-707Google Scholar, 2Ross A.C. FASEB J. 1993; 7: 317-327Crossref PubMed Scopus (170) Google Scholar), represent the drugs of choice for a wide spectrum of severe and recalcitrant skin disorders (12Wieder J.M. Lowe N.J. Dermatol. Clin. 1995; 13: 891-896Abstract Full Text PDF PubMed Google Scholar, 13Lacour M. Mehta-Nikhar B. Atherton D.J. Harper J.I. Br. J. Dermatol. 1996; 134: 1023-1029Crossref PubMed Scopus (121) Google Scholar, 14Plewig G. Albrecht G. Henz B.M. Meigel W. Schöpf E. Stadler R. Hautarzt. 1997; 48: 881-885Crossref PubMed Scopus (11) Google Scholar), and are capable of reversing or suppressing carcinogenesis in many tissues (9Lippman, S. M., Heyman, R. A., Kurie, J. M., Benner, S. E., and Hong, W. K. (1995) J. Cell. Biochem.22, (Suppl.) 1–10Google Scholar, 10Muindi J.R. Cancer Treat. Res. 1996; 870: 305-342Crossref Scopus (11) Google Scholar, 11Sankaranarayanan R. Mathew B. IARC Sci. Publ. 1996; 139: 47-59PubMed Google Scholar).Retinoids act through binding to nuclear receptors that belong to the steroid/thyroid hormone superfamily. It has been proposed that target genes are regulated by these receptors that act as transcription factors activated by retinoids (3DeLuca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (812) Google Scholar, 4van der Saag P.T. Eur. J. Clin. Nutr. 1996; 50: 24-28PubMed Google Scholar, 5Botling J. Castro D.S. Oberg F. Nilsson K. Perlmann T. J. Biol. Chem. 1997; 272: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 6Leid M. Kastner P. Chambon P. Trends Biochem Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar). However, there is also evidence indicating that retinoid action can be mediated through mechanisms not involving the retinoid nuclear receptors (22Chao W.R. Hobbs P.D. Jong L. Zhang X.K. Zheng Y. Wu Q. Shroot B. Dawson M.I. Cancer Lett. 1997; 115: 1-7Crossref PubMed Scopus (44) Google Scholar, 23Hsu C.A. Rishi A.K. Su-Li X. Gerald T.M. Dawson M.I. Schiffer C. Reichert V. Shroot B. Poirer G.C. Fontana J.A. Blood. 1997; 89: 4470-4479Crossref PubMed Google Scholar). In the present study it is demonstrated that the important ribozyme RNase P isolated fromD. discoideum is competitively inhibited by natural and synthetic retinoids.All retinoids tested revealed a dose-dependent inhibition of RNase P activity, indicating that these compounds may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that the type of inhibition of retinol, all-trans retinoic acid, isotretinoin, and acitretin is simply competitive. According to this finding we could assign the potency of inhibitors solely on the basis of the K i value. Thus, acitretin is 184, 2.5, and 1.9 times more potent than retinol, isotretinoin, and all-trans retinoic acid, respectively.An interesting observation is that substitution of the CH3O group for hydrogen in the P position of the aromatic ring of the parent compound (all-trans retinoic acid) (Fig. 1) enhances the inhibitory potency of the retinoid. Because the CH3O group increases the hydrophobicity of acitretin, it is possible that this compound may fit better into hydrophobic site(s) of the RNase P. It has been suggested that the low buoyant density of RNase P in Cs2SO4 gradients (19Stathopoulos C. Kalpaxis D.L. Drainas D. Eur. J. Biochem. 1995; 228: 976-980Crossref PubMed Scopus (31) Google Scholar) can be attributed to the occurrence of an unidentified component such as a fatty acid or lipid. This hypothesis is consistent with the existence of hydrophobic site(s) on RNase P. On the other hand, retinol, which is a very weak inhibitor (K i = 1475 μm), has a hydroxyl group in place of the carboxyl one (Fig. 1), indicating that the absence of the latter affects the binding of the analog with the enzyme. It is also interesting that among the stereoisomers (Fig. 1) all-trans retinoic acid (K i = 15 μm) is stronger inhibitor than isotretinoin (13-cis retinoic acid) (K i = 20 μm), suggesting that the stereochemical properties play an important role in the inhibitory behavior of these molecules.A significant point of interest emerged from the Hill plot analysis (Fig. 6). Only one binding site (n ≅ 1) for retinoids seems to be necessary for the inhibition of RNase P reaction. Because of the kinetic nature of this observation, we cannot exclude the existence of additional retinoid-binding sites on RNase P holoenzyme, which are not involved in the kinetic model of inhibition.Because the retinoids examined in the present study reveal no structural similarity to the substrate (pre-tRNA) of RNase P, the competitive character of the inhibition suggests that these compounds may bind to allosteric inhibition sites of the enzyme. Finally, it is possible that the inhibition of D. discoideum RNase P could have been caused by a retinoid-specific receptor complex; the receptor could have been co-eluted with RNase P activity during the purification procedure. However, this hypothesis can be ruled out, because RNase P activity during the purification through the Sephacryl S-300 column elutes with the void volume of the column, with the enzyme behaving as a protein with very high molecular mass (see “Materials and Methods”), whereas retinoic acid receptors, if present in the extract, will elute in later fractions well apart from the RNase P activity, because of their low molecular mass (<50 kDa) (3DeLuca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (812) Google Scholar). Furthermore, it could be speculated that the inhibition of RNase P activity results from retinoids titrating out a co-activator component from RNase P, for example a bivalent cation like Mg2+; nevertheless, this possibility is definitely ruled out because in this case the inhibition pattern is of partial noncompetitive type. 1A. Tekos, C. Stathopoulos, and D. Drainas, unpublished results. Moreover, other types of RNase P co-activators have not been reported so far. Retinoids, a group of natural and synthetic analogs of vitamin A, play an essential role in vision, growth, and reproduction as well as exhibiting striking effects on cell proliferation, differentiation, and pattern formation during development (1Tsambaos D. Zimmerman B. Roenigk H.H. Maibach H.I. Effects of Synthetic Retinoids on Cellular Systems in Psoriasis. Marcel Dekker Inc., New York1991: 659-707Google Scholar, 2Ross A.C. FASEB J. 1993; 7: 317-327Crossref PubMed Scopus (170) Google Scholar). The discovery that members of the steroid/thyroid hormone receptor superfamily are nuclear retinoic acid-binding proteins tremendously improves our understanding of the mechanisms that mediate the regulatory action of retinoids on gene expression (3DeLuca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (812) Google Scholar, 4van der Saag P.T. Eur. J. Clin. Nutr. 1996; 50: 24-28PubMed Google Scholar, 5Botling J. Castro D.S. Oberg F. Nilsson K. Perlmann T. J. Biol. Chem. 1997; 272: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 6Leid M. Kastner P. Chambon P. Trends Biochem Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar). The retinoid receptors are ligand-activated, DNA-binding, trans-acting, transcription factors (7Beato M. Cell. 1989; 56: 335-344Abstract Full Text PDF PubMed Scopus (2841) Google Scholar, 8Minucci S. Leid M. Toyama R. Saint-Jeannet J.P. Peterson V.J. Horn V. Ishmael J.E. Bhattacharyya N. Dey A. Dawid I.B. Ozato K. Mol. Cell. Biol. 1997; 17: 644-655Crossref PubMed Scopus (148) Google Scholar). Due to their ability to regulate cell differentiation and suppress or reverse the malignant phenotype, retinoids have a potential use as chemopreventive and chemotherapeutic agents in cancers of skin and other organs (9Lippman, S. M., Heyman, R. A., Kurie, J. M., Benner, S. E., and Hong, W. K. (1995) J. Cell. Biochem.22, (Suppl.) 1–10Google Scholar, 10Muindi J.R. Cancer Treat. Res. 1996; 870: 305-342Crossref Scopus (11) Google Scholar, 11Sankaranarayanan R. Mathew B. IARC Sci. Publ. 1996; 139: 47-59PubMed Google Scholar). Moreover, oral synthetic retinoids are presently successfully applied in the management of severe and recalcitrant dermatoses, which were previously regarded as frustrating therapeutic problems (12Wieder J.M. Lowe N.J. Dermatol. Clin. 1995; 13: 891-896Abstract Full Text PDF PubMed Google Scholar, 13Lacour M. Mehta-Nikhar B. Atherton D.J. Harper J.I. Br. J. Dermatol. 1996; 134: 1023-1029Crossref PubMed Scopus (121) Google Scholar, 14Plewig G. Albrecht G. Henz B.M. Meigel W. Schöpf E. Stadler R. Hautarzt. 1997; 48: 881-885Crossref PubMed Scopus (11) Google Scholar). RNase P is a key enzyme in tRNA biogenesis, which cleaves all tRNA precursors endonucleolytically to produce the mature 5′ end. RNase P enzymes are composed of both RNA and protein (15Darr S.C. Brown J.W. Pace N.R. Trends Biochem. Sci. 1992; 17: 178-182Abstract Full Text PDF PubMed Scopus (94) Google Scholar). In vitro, RNA subunits of bacterial enzymes are catalytically active in the absence of protein (16Guerrier-Takada C. Gardiner T. Marsh K. Pace N. Altman S. Cell. 1983; 35: 849-857Abstract Full Text PDF PubMed Scopus (2007) Google Scholar) and are the only known RNA catalysts naturally devoted to act in trans (17Symons R.H. Curr. Opin. Struct. Biol. 1994; 4: 322-330Crossref Scopus (54) Google Scholar). Catalytic activity of RNA subunits, in the absence of protein subunits, has not been demonstrated so far for archaea and eukaryotes RNase P. However, the catalytic center of these RNase P enzymes most likely is associated with the RNA subunits. Eukaryotic RNase P activity has been detected in nuclei, mitochondria, and chloroplasts (15Darr S.C. Brown J.W. Pace N.R. Trends Biochem. Sci. 1992; 17: 178-182Abstract Full Text PDF PubMed Scopus (94) Google Scholar, 18Drainas D. Mol. Biol. Rep. 1996; 22: 135-138Crossref Scopus (7) Google Scholar). Recently, the partial purification and characterization of RNase P from the slime mold Dictyostelium discoideum has been reported (18Drainas D. Mol. Biol. Rep. 1996; 22: 135-138Crossref Scopus (7) Google Scholar,19Stathopoulos C. Kalpaxis D.L. Drainas D. Eur. J. Biochem. 1995; 228: 976-980Crossref PubMed Scopus (31) Google Scholar). In the present study we have examined the effect of natural and synthetic retinoids on RNase P using a cell free system from the slime mold D. discoideum as an in vitro model. D. discoideum RNase P is a nuclear enzyme that has low buoyant density in Cs2SO4 gradients possibly due to the occurrence of an unidentified component such as a fatty acid or lipid (19Stathopoulos C. Kalpaxis D.L. Drainas D. Eur. J. Biochem. 1995; 228: 976-980Crossref PubMed Scopus (31) Google Scholar). This hypothesis is consistent with the existence of hydrophobic site(s) on D. discoideum RNase P and perhaps justifies the role of retinoids as potential inhibitors, being hydrophobic molecules acting in the nucleus. RESULTSThe present data show that all retinoids investigated here (Fig. 1) are capable of inhibiting the RNase P activity of D. discoideum. The substrate for RNase P assays was an in vitro labeled transcript of the S. pombe tRNASer gene supSI (20Drainas D. Zimmerly S. Willis I. Söll D. FEBS Lett. 1989; 251: 84-88Crossref PubMed Scopus (26) Google Scholar).The concentration of all-trans retinoic acid (IC50) at which the product formation is reduced by 50% is equal to 80 μm (Fig. 2,A and B). Although not as effective as all-trans retinoic acid, retinol also inhibited RNase P activity; the IC50 was equal to 500 μm (Fig. 2, C and D). The dose response curves for the synthetic retinoids isotretinoin and acitretin were similar to those observed with the natural retinoids (Fig. 3). The IC50 values were 60 μm (Fig. 3, A and B) and 40 μm (Fig. 3, C and D), respectively. It is obvious from these results that acitretin is the strongest inhibitor among the retinoids tested. It is important to note that RNase P purified through the second DE 52 column was inhibited by retinoids to the same extent as RNase P purified through the S-300 column.The type of inhibition of D. discoideum RNase P activity by the natural and synthetic retinoids was further elucidated by detailed kinetic analysis. Because the yields of RNase P after gel filtration chromatography are low, for the kinetic analysis we used RNase P fractions obtained from DEAE-cellulose chromatography. Due to the high hydrophobicity of retinoids, all assays were carried out in the presence of 10% Me2SO. At this concentration, Me2SO marginally affects (K i = 4m) the catalytic parameters of D. discoideumRNase P. The initial velocity in the presence or absence of retinoids was determined from the initial slopes of time plots (not shown). Fig. 4 shows double reciprocal plots with increasing concentrations of acitretin. The lowest line in Fig. 4represents the data obtained in the absence of inhibitor and Me2SO (control). The calculated apparentK m (K m,app) and the apparent V max (V max,app) values from this plot are 240 nm and 3 pmol/min, respectively. These values are in agreement with previous values reported from our laboratory (19Stathopoulos C. Kalpaxis D.L. Drainas D. Eur. J. Biochem. 1995; 228: 976-980Crossref PubMed Scopus (31) Google Scholar). In the presence of 10% Me2SO the K m,app and theV max,app values are 250 nm and 3 pmol/min, respectively (Fig. 4). The slopes of the lines in Fig. 4 were replotted against the concentration of acitretin, and the results are shown in the top panel of Fig. 4. The linearity of this replot is indicative of simple competitive inhibition and leads to the graphical determination of K i = 8.0 μm from the negative intercept of the line with the I-axis. Further evidence for simple competitive kinetics comes from the Dixon plot, which is shown in Fig. 5. When the slopes of the lines of Fig. 4 are replotted against 1/[pre-tRNA] (Fig. 5, top panel) they give a straight line passing through the origin. This is further evidence of simple competitive inhibition (21Segel I.H. Enzyme kinetics. John Wiley & Sons, Inc., New York1975: 110Google Scholar). Hill plots were obtained by calculating the log[v′/(v 0-v′)] values at each concentration of pre-tRNA and plotting them as a function of logarithmic retinoid concentration. The molecular interaction coefficient (n) for retinoids was given by the slope of these plots. Fig. 6 shows the Hill plot for acitretin obtained at 100 nm pre-tRNA. Then value calculated from this plot is about equal to 1. These results support the notion that the inhibition of D. discoideum RNase P activity by retinoids involves only one retinoid molecule.Figure 4Double reciprocal plot (1/v versus 1/[pre-tRNA]) for RNase P reaction in the presence of acitretin. The reaction was carried out at each one of the indicated concentrations in the presence or in the absence of inhibitor. All reactions were carried out at 37 °C in 20 μl of buffer D in the presence of 10% Me2SO. •, without inhibitor minus Me2SO; ○, without inhibitor plus Me2SO; ▪, with acitretin at 15 μm; ▵, with acitretin at 25 μm; ▴, with acitretin at 30 μm; ▪, with acitretin at 50 μm; ♦, with acitretin at 75 μm. Top panel, replot of the slopes of the double reciprocal lines versus inhibitor (I) concentrations.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Dixon plot for the RNase P reaction in the presence of acitretin. The concentrations of pre-tRNA were as follows: •, 50 nm; ▴, 100 nm; ▵, 125 nm; ■, 150 nm; ▪, 250 nm; ♦, 500 nm. Top panel, replot of the slopes of the Dixon lines versus 1/[pre-tRNA].View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Hill plot of inhibition of the RNase P reaction by acitretin. The molecular interaction coefficient (n) of acitretin was obtained from the slope of this plot. The v′ (in the presence of acitretin) and v 0 (in the absence of acitretin) values were calculated from the corresponding time plots.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The same kinetic examination was carried out for isotretinoin, all-trans retinoic acid, and retinol. All these compounds showed simple competitive inhibition with molecular interaction coefficient equal to 1. The corresponding K i values are given in Table I.Table IEquilibrium constants derived from primary and secondary kinetic plotsCompoundK iμmRetinol1475.0All-trans retinoic acid15.0Isotretinoin20.0Acitretin8.0The K i values are calculated from the negative intercept of the slope replots. Open table in a new tab To address the question of whether retinoids may be capable of affecting also other nucleases, we investigated the effect of retinol, all-trans retinoic acid, isotretinoin, and acitretin on RNase A from bovine pancreas and found that these compounds exert no effect on RNase A activity (data not shown). The present data show that all retinoids investigated here (Fig. 1) are capable of inhibiting the RNase P activity of D. discoideum. The substrate for RNase P assays was an in vitro labeled transcript of the S. pombe tRNASer gene supSI (20Drainas D. Zimmerly S. Willis I. Söll D. FEBS Lett. 1989; 251: 84-88Crossref PubMed Scopus (26) Google Scholar). The concentration of all-trans retinoic acid (IC50) at which the product formation is reduced by 50% is equal to 80 μm (Fig. 2,A and B). Although not as effective as all-trans retinoic acid, retinol also inhibited RNase P activity; the IC50 was equal to 500 μm (Fig. 2, C and D). The dose response curves for the synthetic retinoids isotretinoin and acitretin were similar to those observed with the natural retinoids (Fig. 3). The IC50 values were 60 μm (Fig. 3, A and B) and 40 μm (Fig. 3, C and D), respectively. It is obvious from these results that acitretin is the strongest inhibitor among the retinoids tested. It is important to note that RNase P purified through the second DE 52 column was inhibited by retinoids to the same extent as RNase P purified through the S-300 column. The type of inhibition of D. discoideum RNase P activity by the natural and synthetic retinoids was further elucidated by detailed kinetic analysis. Because the yields of RNase P after gel filtration chromatography are low, for the kinetic analysis we used RNase P fractions obtained from DEAE-cellulose chromatography. Due to the high hydrophobicity of retinoids, all assays were carried out in the presence of 10% Me2SO. At this concentration, Me2SO marginally affects (K i = 4m) the catalytic parameters of D. discoideumRNase P. The initial velocity in the presence or absence of retinoids was determined from the initial slopes of time plots (not shown). Fig. 4 shows double reciprocal plots with increasing concentrations of acitretin. The lowest line in Fig. 4represents the data obtained in the absence of inhibitor and Me2SO (control). The calculated apparentK m (K m,app) and the apparent V max (V max,app) values from this plot are 240 nm and 3 pmol/min, respectively. These values are in agreement with previous values reported from our laboratory (19Stathopoulos C. Kalpaxis D.L. Drainas D. Eur. J. Biochem. 1995; 228: 976-980Crossref PubMed Scopus (31) Google Scholar). In the presence of 10% Me2SO the K m,app and theV max,app values are 250 nm and 3 pmol/min, respectively (Fig. 4). The slopes of the lines in Fig. 4 were replotted against the concentration of acitretin, and the results are shown in the top panel of Fig. 4. The linearity of this replot is indicative of simple competitive inhibition and leads to the graphical determination of K i = 8.0 μm from the negative intercept of the line with the I-axis. Further evidence for simple competitive kinetics comes from the Dixon plot, which is shown in Fig. 5. When the slopes of the lines of Fig. 4 are replotted against 1/[pre-tRNA] (Fig. 5, top panel) they give a straight line passing through the origin. This is further evidence of simple competitive inhibition (21Segel I.H. Enzyme kinetics. John Wiley & Sons, Inc., New York1975: 110Google Scholar). Hill plots were obtained by calculating the log[v′/(v 0-v′)] values at each concentration of pre-tRNA and plotting them as a function of logarithmic retinoid concentration. The molecular interaction coefficient (n) for retinoids was given by the slope of these plots. Fig. 6 shows the Hill plot for acitretin obtained at 100 nm pre-tRNA. Then value calculated from this plot is about equal to 1. These results support the notion that the inhibition of D. discoideum RNase P activity by retinoids involves only one retinoid molecule. The same kinetic examination was carried out for isotretinoin, all-trans retinoic acid, and retinol. All these compounds showed simple competitive inhibition with molecular interaction coefficient equal to 1. The corresponding K i values are given in Table I. The K i values are calculated from the negative intercept of the slope replots. To address the question of whether retinoids may be capable of affecting also other nucleases, we investigated the effect of retinol, all-trans retinoic acid, isotretinoin, and acitretin on RNase A from bovine pancreas and found that these compounds exert no effect on RNase A activity (data not shown). DISCUSSIONThe study of the action of retinoids is of essential importance because these compounds are involved in gene expression, cell growth, and differentiation (1Tsambaos D. Zimmerman B. Roenigk H.H. Maibach H.I. Effects of Synthetic Retinoids on Cellular Systems in Psoriasis. Marcel Dekker Inc., New York1991: 659-707Google Scholar, 2Ross A.C. FASEB J. 1993; 7: 317-327Crossref PubMed Scopus (170) Google Scholar), represent the drugs of choice for a wide spectrum of severe and recalcitrant skin disorders (12Wieder J.M. Lowe N.J. Dermatol. Clin. 1995; 13: 891-896Abstract Full Text PDF PubMed Google Scholar, 13Lacour M. Mehta-Nikhar B. Atherton D.J. Harper J.I. Br. J. Dermatol. 1996; 134: 1023-1029Crossref PubMed Scopus (121) Google Scholar, 14Plewig G. Albrecht G. Henz B.M. Meigel W. Schöpf E. Stadler R. Hautarzt. 1997; 48: 881-885Crossref PubMed Scopus (11) Google Scholar), and are capable of reversing or suppressing carcinogenesis in many tissues (9Lippman, S. M., Heyman, R. A., Kurie, J. M., Benner, S. E., and Hong, W. K. (1995) J. Cell. Biochem.22, (Suppl.) 1–10Google Scholar, 10Muindi J.R. Cancer Treat. Res. 1996; 870: 305-342Crossref Scopus (11) Google Scholar, 11Sankaranarayanan R. Mathew B. IARC Sci. Publ. 1996; 139: 47-59PubMed Google Scholar).Retinoids act through binding to nuclear receptors that belong to the steroid/thyroid hormone superfamily. It has been proposed that target genes are regulated by these receptors that act as transcription factors activated by retinoids (3DeLuca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (812) Google Scholar, 4van der Saag P.T. Eur. J. Clin. Nutr. 1996; 50: 24-28PubMed Google Scholar, 5Botling J. Castro D.S. Oberg F. Nilsson K. Perlmann T. J. Biol. Chem. 1997; 272: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 6Leid M. Kastner P. Chambon P. Trends Biochem Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar). However, there is also evidence indicating that retinoid action can be mediated through mechanisms not involving the retinoid nuclear receptors (22Chao W.R. Hobbs P.D. Jong L. Zhang X.K. Zheng Y. Wu Q. Shroot B. Dawson M.I. Cancer Lett. 1997; 115: 1-7Crossref PubMed Scopus (44) Google Scholar, 23Hsu C.A. Rishi A.K. Su-Li X. Gerald T.M. Dawson M.I. Schiffer C. Reichert V. Shroot B. Poirer G.C. Fontana J.A. Blood. 1997; 89: 4470-4479Crossref PubMed Google Scholar). In the present study it is demonstrated that the important ribozyme RNase P isolated fromD. discoideum is competitively inhibited by natural and synthetic retinoids.All retinoids tested revealed a dose-dependent inhibition of RNase P activity, indicating that these compounds may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that the type of inhibition of retinol, all-trans retinoic acid, isotretinoin, and acitretin is simply competitive. According to this finding we could assign the potency of inhibitors solely on the basis of the K i value. Thus, acitretin is 184, 2.5, and 1.9 times more potent than retinol, isotretinoin, and all-trans retinoic acid, respectively.An interesting observation is that substitution of the CH3O group for hydrogen in the P position of the aromatic ring of the parent compound (all-trans retinoic acid) (Fig. 1) enhances the inhibitory potency of the retinoid. Because the CH3O group increases the hydrophobicity of acitretin, it is possible that this compound may fit better into hydrophobic site(s) of the RNase P. It has been suggested that the low buoyant density of RNase P in Cs2SO4 gradients (19Stathopoulos C. Kalpaxis D.L. Drainas D. Eur. J. Biochem. 1995; 228: 976-980Crossref PubMed Scopus (31) Google Scholar) can be attributed to the occurrence of an unidentified component such as a fatty acid or lipid. This hypothesis is consistent with the existence of hydrophobic site(s) on RNase P. On the other hand, retinol, which is a very weak inhibitor (K i = 1475 μm), has a hydroxyl group in place of the carboxyl one (Fig. 1), indicating that the absence of the latter affects the binding of the analog with the enzyme. It is also interesting that among the stereoisomers (Fig. 1) all-trans retinoic acid (K i = 15 μm) is stronger inhibitor than isotretinoin (13-cis retinoic acid) (K i = 20 μm), suggesting that the stereochemical properties play an important role in the inhibitory behavior of these molecules.A significant point of interest emerged from the Hill plot analysis (Fig. 6). Only one binding site (n ≅ 1) for retinoids seems to be necessary for the inhibition of RNase P reaction. Because of the kinetic nature of this observation, we cannot exclude the existence of additional retinoid-binding sites on RNase P holoenzyme, which are not involved in the kinetic model of inhibition.Because the retinoids examined in the present study reveal no structural similarity to the substrate (pre-tRNA) of RNase P, the competitive character of the inhibition suggests that these compounds may bind to allosteric inhibition sites of the enzyme. Finally, it is possible that the inhibition of D. discoideum RNase P could have been caused by a retinoid-specific receptor complex; the receptor could have been co-eluted with RNase P activity during the purification procedure. However, this hypothesis can be ruled out, because RNase P activity during the purification through the Sephacryl S-300 column elutes with the void volume of the column, with the enzyme behaving as a protein with very high molecular mass (see “Materials and Methods”), whereas retinoic acid receptors, if present in the extract, will elute in later fractions well apart from the RNase P activity, because of their low molecular mass (<50 kDa) (3DeLuca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (812) Google Scholar). Furthermore, it could be speculated that the inhibition of RNase P activity results from retinoids titrating out a co-activator component from RNase P, for example a bivalent cation like Mg2+; nevertheless, this possibility is definitely ruled out because in this case the inhibition pattern is of partial noncompetitive type. 1A. Tekos, C. Stathopoulos, and D. Drainas, unpublished results. Moreover, other types of RNase P co-activators have not been reported so far. The study of the action of retinoids is of essential importance because these compounds are involved in gene expression, cell growth, and differentiation (1Tsambaos D. Zimmerman B. Roenigk H.H. Maibach H.I. Effects of Synthetic Retinoids on Cellular Systems in Psoriasis. Marcel Dekker Inc., New York1991: 659-707Google Scholar, 2Ross A.C. FASEB J. 1993; 7: 317-327Crossref PubMed Scopus (170) Google Scholar), represent the drugs of choice for a wide spectrum of severe and recalcitrant skin disorders (12Wieder J.M. Lowe N.J. Dermatol. Clin. 1995; 13: 891-896Abstract Full Text PDF PubMed Google Scholar, 13Lacour M. Mehta-Nikhar B. Atherton D.J. Harper J.I. Br. J. Dermatol. 1996; 134: 1023-1029Crossref PubMed Scopus (121) Google Scholar, 14Plewig G. Albrecht G. Henz B.M. Meigel W. Schöpf E. Stadler R. Hautarzt. 1997; 48: 881-885Crossref PubMed Scopus (11) Google Scholar), and are capable of reversing or suppressing carcinogenesis in many tissues (9Lippman, S. M., Heyman, R. A., Kurie, J. M., Benner, S. E., and Hong, W. K. (1995) J. Cell. Biochem.22, (Suppl.) 1–10Google Scholar, 10Muindi J.R. Cancer Treat. Res. 1996; 870: 305-342Crossref Scopus (11) Google Scholar, 11Sankaranarayanan R. Mathew B. IARC Sci. Publ. 1996; 139: 47-59PubMed Google Scholar). Retinoids act through binding to nuclear receptors that belong to the steroid/thyroid hormone superfamily. It has been proposed that target genes are regulated by these receptors that act as transcription factors activated by retinoids (3DeLuca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (812) Google Scholar, 4van der Saag P.T. Eur. J. Clin. Nutr. 1996; 50: 24-28PubMed Google Scholar, 5Botling J. Castro D.S. Oberg F. Nilsson K. Perlmann T. J. Biol. Chem. 1997; 272: 9443-9449Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 6Leid M. Kastner P. Chambon P. Trends Biochem Sci. 1992; 17: 427-433Abstract Full Text PDF PubMed Scopus (803) Google Scholar). However, there is also evidence indicating that retinoid action can be mediated through mechanisms not involving the retinoid nuclear receptors (22Chao W.R. Hobbs P.D. Jong L. Zhang X.K. Zheng Y. Wu Q. Shroot B. Dawson M.I. Cancer Lett. 1997; 115: 1-7Crossref PubMed Scopus (44) Google Scholar, 23Hsu C.A. Rishi A.K. Su-Li X. Gerald T.M. Dawson M.I. Schiffer C. Reichert V. Shroot B. Poirer G.C. Fontana J.A. Blood. 1997; 89: 4470-4479Crossref PubMed Google Scholar). In the present study it is demonstrated that the important ribozyme RNase P isolated fromD. discoideum is competitively inhibited by natural and synthetic retinoids. All retinoids tested revealed a dose-dependent inhibition of RNase P activity, indicating that these compounds may have a direct effect on tRNA biogenesis. Detailed kinetic analysis showed that the type of inhibition of retinol, all-trans retinoic acid, isotretinoin, and acitretin is simply competitive. According to this finding we could assign the potency of inhibitors solely on the basis of the K i value. Thus, acitretin is 184, 2.5, and 1.9 times more potent than retinol, isotretinoin, and all-trans retinoic acid, respectively. An interesting observation is that substitution of the CH3O group for hydrogen in the P position of the aromatic ring of the parent compound (all-trans retinoic acid) (Fig. 1) enhances the inhibitory potency of the retinoid. Because the CH3O group increases the hydrophobicity of acitretin, it is possible that this compound may fit better into hydrophobic site(s) of the RNase P. It has been suggested that the low buoyant density of RNase P in Cs2SO4 gradients (19Stathopoulos C. Kalpaxis D.L. Drainas D. Eur. J. Biochem. 1995; 228: 976-980Crossref PubMed Scopus (31) Google Scholar) can be attributed to the occurrence of an unidentified component such as a fatty acid or lipid. This hypothesis is consistent with the existence of hydrophobic site(s) on RNase P. On the other hand, retinol, which is a very weak inhibitor (K i = 1475 μm), has a hydroxyl group in place of the carboxyl one (Fig. 1), indicating that the absence of the latter affects the binding of the analog with the enzyme. It is also interesting that among the stereoisomers (Fig. 1) all-trans retinoic acid (K i = 15 μm) is stronger inhibitor than isotretinoin (13-cis retinoic acid) (K i = 20 μm), suggesting that the stereochemical properties play an important role in the inhibitory behavior of these molecules. A significant point of interest emerged from the Hill plot analysis (Fig. 6). Only one binding site (n ≅ 1) for retinoids seems to be necessary for the inhibition of RNase P reaction. Because of the kinetic nature of this observation, we cannot exclude the existence of additional retinoid-binding sites on RNase P holoenzyme, which are not involved in the kinetic model of inhibition. Because the retinoids examined in the present study reveal no structural similarity to the substrate (pre-tRNA) of RNase P, the competitive character of the inhibition suggests that these compounds may bind to allosteric inhibition sites of the enzyme. Finally, it is possible that the inhibition of D. discoideum RNase P could have been caused by a retinoid-specific receptor complex; the receptor could have been co-eluted with RNase P activity during the purification procedure. However, this hypothesis can be ruled out, because RNase P activity during the purification through the Sephacryl S-300 column elutes with the void volume of the column, with the enzyme behaving as a protein with very high molecular mass (see “Materials and Methods”), whereas retinoic acid receptors, if present in the extract, will elute in later fractions well apart from the RNase P activity, because of their low molecular mass (<50 kDa) (3DeLuca L.M. FASEB J. 1991; 5: 2924-2933Crossref PubMed Scopus (812) Google Scholar). Furthermore, it could be speculated that the inhibition of RNase P activity results from retinoids titrating out a co-activator component from RNase P, for example a bivalent cation like Mg2+; nevertheless, this possibility is definitely ruled out because in this case the inhibition pattern is of partial noncompetitive type. 1A. Tekos, C. Stathopoulos, and D. Drainas, unpublished results. Moreover, other types of RNase P co-activators have not been reported so far. We thank Dr. D. L. Kalpaxis for critical reading of the manuscript.
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