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Ornithine decarboxylase antizyme: a novel type of regulatory protein
I
TIBS 21 - JANUARY 1996
REVIEWS
inhibited by cycioheximide but not by actinomycin D, suggesting that antiz3ane is the protein involved in the polyamine-induced destabilization of ODC. In order to examine this possibility, changes in the cellular amount of ODC-antizyme complex in the presence and absence of exogenous polyamines were analysed using two replacement assay methods 15,16. In these assays, either active ODC is released from the complex by replacement with excess amounts of ODC, or antizyme is irreversibly inactivated by difluoromethylornithine (DFMO) or by an antizyme inhibitor. This inhibitor is a potential regulatory protein that binds to antiAntizyme plays an important regulatory role in the synthesis of ornithine zyme with higher affinity than that of decarboxylase (ODC), a key enzyme of polyamine synthesis in higher ODC17,18. The results of these assays animals. As well as inactivating polyamine uptake, antizyme is induced were that, in hepatoma tissue culture by polyamine-enhanced translational frameshifting, and binds to ODC, (HTC) cells, the cellular level of accelerating its ATP-dependent degradation, a process catalysed by the ODC-antizy~ne complex increased after 26S proteasome. ODC activity passed a peak, reaching its own peak several hours later. The peak level of the complex was less than one POLYAMINES SUCH AS putrescine have been revealed, but further details tenth of the peak ODC level, suggesting that the antizyme-induced inhibition of (also known as diamine), spermidine can be found in Refs 7, 8. ODC activity does not have much and spermine are essential for cell significance in ODC regulation. There Polyamine repression of ODC by antizymegrowth and, in some cases, cell dip was a good correlation between the ferentiation 1. Ornithine decarboxylase dependent destabilization ODC is under strict negative-feed- antizyme:ODC ratio and the rate of (ODC), a key enzyme for the synthesis of polyamines, is induced dramatically back control by its polyamine products ODC decay (the reciprocal of half-lif@ 6. by various growth stimuli z, suggesting in order to protect cells from an over- In addition, a marked accumulation of that the enzyme might be a proto- accumulation of polyamines that might ODC-antizyme complex was observed oncogene product 3,4. It turns over cause c~otoxicity or cellular transfor- in ODC-stabilized HMOA cells ~s. These rapidly, with a variable half-life ranging mation. Three mechanisms have been observations led to a hypothesis that from several minutes (the shortest proposed for the repression of ODC by antiz3une cycling stimulates ODC degrahalf-life among cellular proteins) to polyamines: (1) translational suppres- dation. This recycling is a prerequisite more than one hour, and it. is rapidly sion of ODC synthesis 9, (2) acceleration for ODC inhibition, as a small amount of suppressed by the accumulation of of ODC degradation ml, and (3) induc- antizyme can promote the degradation tion of antizyme, an ODC-inhibitory of a much larger amount of ODC in the cellular polyamines 5,6. ODC antizyme was originally found protein m3. Exogenously added poly- presence of protein synthesis inhibitor. as an ODC-inhibitory protein induced amines cause the decay of ODC acti~ty by polyamines. Recently, it has been in a characteristic manner that begins Antizyme degradation of ODC can be found that antizyme stimulates energy- after a short lag-period and proceeds independent of pelyamines Recent studies in this and other la~ dependent degradation of ODC, a pro- with an increasing rate until the rate of cess catalysed by the 26S proteasome. decay is faster than that with cyclo- oratories have proved the above workAntizyme also suppresses cellular heximide m~. This decay is abolished ing hypothesis owing to the develop uptake of polyamines, but conversely, it by simultaneous addition of cyclo- ment of gene technology. A 5'-truncated is induced by the unusual mechanism heximide H,~4 but not actinomycin D rat antizyme cDNA ~1) was inserted of translational frameshifting caused by (Ref. 14). These results suggest that a into an expression vector at a site polyamine accumulation. Thus, anti- protein induced by polyamines at a under the control of a glucocorticoidzyme plays a pivotal role in a unique post-transcriptional level stimulates inducible promoter and introduced into HTC cells. The addition of dexamethanegative feedback system that prevents ODC degradation. sone, a synthetic glucocorticoid, to excess accumulation of cellular polyamines by both accelerating ODC Antizyme recycles to promote ODC stable transfected HZ7 cells, increased the amount of Zl mRNA and induced degradation and suppressing polyamine degradation Antizyme was discovered in 1976 active antizyme in the absence of uptake. This review will briefly summarize how these unique mechanisms by Canellakis and co-workers as a exogenous polyamines. Addition of dexapolyamine-inducible ODC inhibitor in methasone to cells with high levels of rat liver and several lines of cultured pre-induced ODC elicited a rapid decay S. Hayashi,Y. Murakami and S. Matsufuji are cells 12,13. It is a 26.5kDa protein that of ODC after a short lag time, which at the Departmentof BiochemistryII, The can reversibly bind to ODC. The induc- could be prevented with cycloheximide. Jikei UniversitySchoolof Medicine,Minato-ku, tion of antizyine by polyamines is These results were similar to those Tokyo105, Japan.
Ornithine decarboxylase antizyme: a novel type of regulatory protein
Shin-ichi Hayashi, Yasuko Murakami and Senya Matsufuji
9 1996,ElsevierScienceLtd
27
REVIEWS
TIBS 2 1 - JANUARY 1996
ODC dimer
Omithine
Figure 1 Antizyme-mediated negative feedback regulation of ornithine decarboxylase (ODC) and polyamine uptake. Active ODC comprises two identical monomer subunits with two active sites formed at the interface. The enzymatically active dimeric form of ODC is in rapid equilibrium with the inactive monomeric form. Antizyme (AZ), which is induced by translational frameshifting, preferentially binds with an inactive ODC monomer to form an ODC-antizyme complex. This binding causes a conformational change of the ODC subunit, exposing the carboxy-terminal region to attack from the 26S proteasome complex. ODC is broken down by the proteasome to a mixture of peptides of 5-11 amino acid residues, whereas antizyme is released and recycled to destabilize more ODC monomers. Antizyme also inactivates polyamine uptake at the cell membrane, and is bound by an antizyme inhibitor that has a higher affinity for antizyme than ODC does, and apparently stabilizes ODC by reducing the amount of antizyme available to ODC destabilization.
observed upon addition of polyamines, and clearly indicate that antizyme accelerates ODC degradation in whole cells independently of polyamines, and that the polyamine effect to elicit ODC decay is mediated mostly, if not totally, by antizyme ~9. Further evidence was obtained in studies with an in vitro ODC-degradation system. Kahana and co-workers developed a reticulocyte lysate system in which ODC synthesized in vitro was degraded in the presence of ATP2~ In their system, however, exogenously added ODC was not degraded, leaving some doubt as to the authenticity of the system. In cell-free extracts from both Chinese hamster ovary (CHO) cells and HTC cells, exogenously or endogenously labeled ODC is degraded in the presence of ATP and antizym e21. Omission of either ATP or antizyme results in a marked suppression of ODC degradation. The rate of ODC degradation is accelerated by increasing the amount of antizyme to a maximum when the antizyme to ODC ratio is about 1:1. It was also demonstrated that Kahana's system reflects ODC degradation in whole cells; the reticulocyte lysate system contains a very small amount of endogenous antizyme, which is sufficient for degradation of small amounts of endogenous ODC, but not for relatively large amounts of
28
exogenous ODe 22. Li and Coffino observed that decay of ODC synthesized in vitro was stimulated by in vitro-synthesized antizyme in reticulocyte lysates 23.
Involvement of the protease Three major groups of proteases are
known to be present in cells; lysosomal cathepsins 24, Ca2§ cytosolic calpains 25 and proteasomes 2e. Inhibitor studies have led to the suggestion that proteasomes are the most probable candidates to be involved in ODC degradation. In collaboration with Tanaka's group from Tokushima University, we demonstrated that ODC-degrading activity is completely inhibited by treatment with an anti-proteasome antibody27. Two kinds of proteasomes are known to be present in the cytosol, namely the 20S proteasome made up of 15-20 different subunits, and the 26S proteasome, which consists of the 20S proteasome core associated with 15-20 regulatory subunits 2~. In a highly purified fraction of 26S proteasome (isolated on a glycerol density gradient), ODC-degrading activity exhibited a pattern identical to the proteolytic activities of the 26S proteasome 27, which was of particular interest because this proteasome was thought to degrade poly-ubiquitinated proteins only. However, ODC is well degraded in
a reconstituted system consisting of purified 26S proteasome, antizyme and ATP, but lacking ubiquitin or related enzymes. This is the first case of a nonubiquitinated protein being degraded by the 26S proteasome, and the first example of the in vitro degradation of a cellular protein by a specific ATPdependent protease. ATP is required for both ODC degradation and for the association of the 20S proteasome with regulatory subunits to form the 26S proteasome. Here, ATP is required as an energy source since non-metabolizable ATP analogs cannot be used instead 27. However, the mechanism for this ATP requirement remains to be clarified.
Structural considerationsof ODC Mouse ODC consists of two identical subunits of 461 amino acids. Two active sites formed with amino acids from both subunits are present at the interface. The active ODC homodimer is in equilibrium with the inactive monomer, to which antizyme preferentially binds 2s (see Fig. 1). Structural elements of ODC responsible for its degradation have been pursued by Coffino and co-workers who used various deletion mutations and chimeric variants of ODC. The carboxy-terminal region of ODC (amino acids 423-461), which is missing in stable trypanosomal ODC, is essential for both antizyme-dependent and
REVIEWS
T I B S 2 1 - JANUARY 1 9 9 6
constitutive degradations of ODe 23'29. The region contains one of two PEST regions in mouse ODC (amino acids 423--449). PEST regions are rich in proline (P), glutamic acid (E), serine (S) and threonine (T), and are thought to be a signal for selective proteotysis (PEST hypothesis3~ In addition, a single amino acid exchange in this carboxyterminal region (Cys441 to Try441) results in stable ODC in HMOA cells 31. Furthermore, a region of mouse ODC (amino acids 117-140) is needed for binding with antizyme, whereas another internal PEST region (amino acids 298--333) appears not to be associated with ODC instability.
Antizyme functional domain Coffino's group have shown that the carboxy-terminal half of antizyme (amino acids 106-212) alone can bind with ODC and inactivate it, but an adjacent region (amino acids 55-105) is necessary for destabilizing ODC33. Our own experiments agreed with this result32. These observations suggest that the binding of antizyme to an ODC subunit not only prevents reassociation of inactive subunits, but also causes a conformational change of the subunit. This results in exposure of the ODC carboxy-terminal region to attack from the 26S proteasome. Most antizyme molecules are then liberated intact and recycled to destabilize another ODC subunit (Fig. 1). Chromatographic analysis of the ODC degradation products showed that it is broken down into many different
peptides of 5-11 amino acids. However, free amino acids were not detected, indicating that ODC is degraded by endoproteolysis34. This could have implications for the participation of the 26S proteasome in the processing of class I major histocompatibility complex (MHC) antigenic peptides.
Polyamine-stimulated translational frameshifting As described earlier, induction of antizyme is not dependent on mRNA synthesis. Northern blot analysis showed that antizyme mRNA is present in substantial amounts in all rat tissues examined, either with or without polyamine treatment35. Consistent with this, the antizyme gene has a potent promoter activity36 and antizyme mRNA has a long half-life (12 hours) 35. When full-length cDNAs and a genomic clone36 of rat antizyme were sequenced, an unusual structure for antizyme mRNA37 was revealed. Close to the 5' end is an open reading frame (ORF1) that is too short to encode antizyme. A long overlapping ORF (ORF2), containing the +1 frame with respect to ORF1, lacks an AUG initiator codon, although this frame was used for the expression of Zl antizyme in transfected HZ7 cells using an artificial initiator. The discontinuity of the reading frames is conserved in both human38 and frog39 antizymes. Therefore, an unusual mechanism involving either translational frameshifting or RNA editing, is needed to translate antizyme mRNA. Ample production of
full-length antizyme was observed when a variant antizyme mRNA, lacking a single nucleotide at the termination codon of ORF1, was translated using a rabbit reticulocyte lysate system. By contrast, there was hardly any translation of wild-type antizyme mRNA in the absence of polyamines. Addition of spermidine stimulated translation in a dose-dependent manner with an optimum effect at 0.6raM. The mRNA sequence recovered from the lysate after translation remained unchanged, thus excluding the possibility that the event is a result of RNA editing. Therefore, these results indicate that polyamines induce antizyme by stimulating programmed translational frameshifting4~ Rom and Cahana obtained similar results with antizyme cDNA4~ and the amino acid sequence at the frameshift site was determined using a reporter system. The UGA termination signal and a pseudoknot structure were also identified as downstream stimulators. Although an increasing number of programmed frameshifts have been found to be involved in the gene expression of eukaryotic viruses, the antizyme gene is the first eukaryotic cellular gene known to require frameshifting for its expression. Also, this is only the second reported case of programmed frame~ shifting used for regulation, the first being bacterial polypeptide chain release factor-2. The detailed molecular mechanism, in particular, how polyamines stimulate the frameshift, remains to be clarified by future studies.
Table I. ODC characteristics in different organisms ODC structure
Turnover rate of ODC
Organism
Identity in antizymebinding region a
Carboxy-terminal region b
PEST~ region
- Polyamine
+ Polyamine
Presence of antizyme
Mouse
24
0
+
Moderate
Rapid
+
Chicken
18
0
+
Moderate
Rapid
+
Xenopus
20
0
+
Moderate
Rapid
+
9
-49
?
ND
ND
ND
Saccharomyces cerevisiae
13
-48
+
Moderate
Moderate
Neurospora crassa
11
-41
+
Slow
Moderate
6
-28
+
Slow
Slow
12
-36
-
Slow
Slow
Drosophila
Leishmania Trypanosoma
ND
aNumber of amino acids identical to antizyme-binding region of mouse ODC, which is composed of 24 amino acids. bNumber of deleted amino acids compared with mouse ODC. CRich in proiine, glutamic acid, serine and threonine. Putative signals for selective proteolysis. ND, not determined.
29
REVIEWS
TIBS 21 - JANUARY 1996
Antizyme regulates polyamlne levels
and also has a structure that requires 18 Murakami, Y., Matsufuji, S., Nishiyama, M. and Hayashi, S. (1989) Biochem. J. 259, 839-845 frameshifting for translation 4~ Rapid 19 Murakami, Y., Matsufuji, S., Miyazaki, Y. and ODC turnover and antizyme-mediated Hayashi, S. (1992) J. Biol. Chem. 267, polyamines. Mitchell and co-workers feedback regulation also appear to 13138-13141 found that the involvement of an operate in insect cells (S. Hayashi, 20 Bercovich,Z., Rosenberg-Hasson,Y., Ciechanover,A. and Kahana, C. (1989) J. Bio/. uncharacterized short-lived protein unpublished). Among lower eukaryotes, Chem. 264, 15949-15952 induced by polyamines is essential in such as Saccharomyces and Neurospora, 21 Murakami, Y. et aL (1992) Biochem. J. 283, this process. In their DFMO-resistant ODC turns over moderately rapidly and 661-664 variant cell line derived from HTC cells, antizyme is not detected. In parasites, 22 Murakami, Y. eta/. (1993) Biochem../. 295, 305-308 both ODC and polyamine transporP 2 such as Trypanosoma and Leishmania, 23 Li, X. and Coffino, P. (1993) Mo/. Cell. Biol. 13, are stable and neither are subject to ODC is very stable and antizyme is 2377-2383 feedback regulation by polyamines. not present. Although the presence of 24 Saido, T. C., Sofimachi, H. and Suzuki, K. (1994) FASEB./. 8, 814-822 Recently, it was found that the trans- antizyme-like proteins has been reT. et al. (1993) J. Struct. Biol. 111, fected antizyme gene was induced by ported in prokaryotes and plants, of 25 Yoshimura, 200-211 dexamethasone-suppressed polyamine which some have been well character- 26 Ciechanover,A. (1994) Cell 79, 13-21 transport in HZ7 ceils 43. In collabo- ized 46,47,their physiological roles remain 27 Murakami, Y. et al. (1992) Nature 360, 597-599 28 Mitchell, J. L. A. and Chen, H. J. (1990) ration with lgarashi's group from Chiba to be clarified by future studies. Biochim. Biophys. Acta 1037, 115-121 University, we obtained the same 29 Li, X. and Coffino, P. (1992) Mol. Cell. Biol. 12, results ~. Thus, antizyme effectively 3356-3362 References 30 Rechsteiner, M. (1988) Adv. Enz. Regul. 27, suppresses cellular polyamine levels by 1 Tabor,C. W. and Tabor, H. (1984) Annu. Rev. 135-151 both destabilizing ODC and inactivating Biochem. 53, 749-790 31 Miyazaki, Y., Matsufuji, S., Murakami, Y. and 2 Scalabrino, G. (1994) Curr. Trends Exp. the polyamine uptake system (Fig. 1). Hayashi, S. (1993) Eur. J. Biochem. 214, Endocrinol. 2, 153-183 It would be exciting to find that dis837-844 3 Auvinen, M., Paasinen, A., Anderson, A. C. and 32 Li, X. and Coffino, Y. (1994) Mo/. Cell Bio/. 14, ruption of antizyme function is related HOItt~, E. (1992) Nature 360, 355-358 87-92 4 Tamori, A. et al. (1995) Cancer Res. 55, to human disease. As mentioned before, 33 Ichiba, T. et al. (1994) Biochem. Biophys. Res. 3500-3505 ODC is now considered to be a possible Commun. 200, 1721-1727 5 Hayashi, S. (1989) in Ornithine Decarboxylase: proto-oncogene product 3,4 and there34 Tokunaga,F. et al. (1994) J. Biol. Chem. 269, Biology, Enzymolof~y,and Molecular Genetics 17382-17385 fore, antizyme might act as a tumor sup(Hayashi, S., ed.), pp. 35-45, PergamonPress 35 Matsufuji, S. eta/. (1990) J. Biochem. 108, 6 Murakami, Y., Matsufuji, S., Miyazaki, Y. and pressor. This possibility will be tested 365-371 Hayashi, S. (1994) Biochem. J. 304, 183-187 by using chromosomal mapping and by 36 Miyazaki, Y., Matsufuji, S. and Hayashi, S. 7 Hayashi, S. and Canellakis, E. S. (1989) in deactivating the antizyme gene. (1992) Gene 113, 191-197 Omithine Decarboxylase, Biology, Enzymolog)/, Polyamine transport is known to be under negative feedback control by
and Molecular Genetics (Hayashi, S., ed.),
Concludingremarks The regulation of ODC degradation has many unique features, such as the extreme rapidity of ODC degradation, ubiquitin-independent degradation by the 26S proteasome, frameshift-dependent translational regulation and the dual action of antizyme. It would be intriguing to discover how such a unique regulatory system has been developed during biological evolution. A comparative biochemical approach should give us some hints on that question (Table I). So far, the rapid antizymedependent turnover of ODC has been observed in mammals, avians (chicken) and amphibians (frogs). Frog antizyme mRNA has 65% identity to rat mRNA,
pp. 47-58, PergamonPress 8 Hayashi, S. and Murakami, Y. (1995) Biochem. J. 306, 1-10 9 L6vkvist, E., Strejnborg, L. and Persson, L. (1993) Eur. J. Biochem. 215, 753-759 10 Kanamoto, R., Utsunomiya, K., Kameji, T. and Hayashi, S. (1986) Eur. J. Biochem. 154, 539-544 11 H61tt~, E. and Pohjanpelto, P. (1986) J. Biol. Chem. 261, 9502-9508 12 Fong,W. F., Heller, J. S. and Canellakis, E. S. (1976) Biochim. Biophys. Acta 428, 456-465 13 Heller, J. S., Fong, L. S. and Canellakis, E. S. (1976) Proc. Natl Acad. Sci. USA 73, 1358-1362 14 Mitchell, J. L. A., Mahan, D. W., McCann, P. P. and Qasba, P. (1985) Biochim. Biophys. Acta 840, 309-316 15 Murakami, Y., Fujita, K., Kameji, T. and Hayashi, S. (1985) Biochem J. 225, 689-697 16 Murakami, Y. and Hayashi, S. (1985) Biochem. J. 226, 893-896 17 Fujita, K., Murakarni, Y. and Hayashi, S. (1982) Biochem. J. 204, 647-652
37 Matsufuji, S. et aL (1995) Cell 80, 51-60 38 Tewafi, D. S. eta/. (1994) Biochim. Biophys. Acta 1209, 293-295 39 Ichiba, T., Matsufuji, S., Miyazaki, Y. and Hayashi, S. (1995) Biochim. Biophys. Acta
1262, 83-86 40 Gesteland, R. F., Weiss, R. 8. and At~ns, J. F. (1992) Science 257, 1640-1641 41 Rom, E. and Cahana, C. (1994) Proc. Natl Acad. Sci. USA 91, 3959-3963. Correction: Proc. Nat/ Acad. Sci. USA 91, 9195 42 Mitchell, J. L. A., Hoff, J. A. and BareyaI-Leyser,A. (1991) Arch. Biochem. Biophys. 290, 143-152 43 Mitchell, J. L. A., Diveley,R. R. and BareyalLeyser, A. (1992) Biochim. Biophys. Acta 1136,
136-142 44 Mitchell, J. L. A., Judd, G. G., BareyaI-Leyser,A. and Ling, S. Y. (1994) Biochem. J. 299, 19-22 45 Suzuki, T. et al. (1994) Proc. Nat/Acad. Sci. USA 91, 8930--8934 46 Panagiotidis, C. A. and Canellakis, E. S. (1984) J. Biol. Chem. 259, 15025-15027 47 Canellakis, E. S. et al. (1993) Proc. Nat/Acad. ScL USA 90, 7129-7133
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TIBS 21 - JANUARY 1996
REVIEWS
inhibited by cycioheximide but not by actinomycin D, suggesting that antiz3ane is the protein involved in the polyamine-induced destabilization of ODC. In order to examine this possibility, changes in the cellular amount of ODC-antizyme complex in the presence and absence of exogenous polyamines were analysed using two replacement assay methods 15,16. In these assays, either active ODC is released from the complex by replacement with excess amounts of ODC, or antizyme is irreversibly inactivated by difluoromethylornithine (DFMO) or by an antizyme inhibitor. This inhibitor is a potential regulatory protein that binds to antiAntizyme plays an important regulatory role in the synthesis of ornithine zyme with higher affinity than that of decarboxylase (ODC), a key enzyme of polyamine synthesis in higher ODC17,18. The results of these assays animals. As well as inactivating polyamine uptake, antizyme is induced were that, in hepatoma tissue culture by polyamine-enhanced translational frameshifting, and binds to ODC, (HTC) cells, the cellular level of accelerating its ATP-dependent degradation, a process catalysed by the ODC-antizy~ne complex increased after 26S proteasome. ODC activity passed a peak, reaching its own peak several hours later. The peak level of the complex was less than one POLYAMINES SUCH AS putrescine have been revealed, but further details tenth of the peak ODC level, suggesting that the antizyme-induced inhibition of (also known as diamine), spermidine can be found in Refs 7, 8. ODC activity does not have much and spermine are essential for cell significance in ODC regulation. There Polyamine repression of ODC by antizymegrowth and, in some cases, cell dip was a good correlation between the ferentiation 1. Ornithine decarboxylase dependent destabilization ODC is under strict negative-feed- antizyme:ODC ratio and the rate of (ODC), a key enzyme for the synthesis of polyamines, is induced dramatically back control by its polyamine products ODC decay (the reciprocal of half-lif@ 6. by various growth stimuli z, suggesting in order to protect cells from an over- In addition, a marked accumulation of that the enzyme might be a proto- accumulation of polyamines that might ODC-antizyme complex was observed oncogene product 3,4. It turns over cause c~otoxicity or cellular transfor- in ODC-stabilized HMOA cells ~s. These rapidly, with a variable half-life ranging mation. Three mechanisms have been observations led to a hypothesis that from several minutes (the shortest proposed for the repression of ODC by antiz3une cycling stimulates ODC degrahalf-life among cellular proteins) to polyamines: (1) translational suppres- dation. This recycling is a prerequisite more than one hour, and it. is rapidly sion of ODC synthesis 9, (2) acceleration for ODC inhibition, as a small amount of suppressed by the accumulation of of ODC degradation ml, and (3) induc- antizyme can promote the degradation tion of antizyme, an ODC-inhibitory of a much larger amount of ODC in the cellular polyamines 5,6. ODC antizyme was originally found protein m3. Exogenously added poly- presence of protein synthesis inhibitor. as an ODC-inhibitory protein induced amines cause the decay of ODC acti~ty by polyamines. Recently, it has been in a characteristic manner that begins Antizyme degradation of ODC can be found that antizyme stimulates energy- after a short lag-period and proceeds independent of pelyamines Recent studies in this and other la~ dependent degradation of ODC, a pro- with an increasing rate until the rate of cess catalysed by the 26S proteasome. decay is faster than that with cyclo- oratories have proved the above workAntizyme also suppresses cellular heximide m~. This decay is abolished ing hypothesis owing to the develop uptake of polyamines, but conversely, it by simultaneous addition of cyclo- ment of gene technology. A 5'-truncated is induced by the unusual mechanism heximide H,~4 but not actinomycin D rat antizyme cDNA ~1) was inserted of translational frameshifting caused by (Ref. 14). These results suggest that a into an expression vector at a site polyamine accumulation. Thus, anti- protein induced by polyamines at a under the control of a glucocorticoidzyme plays a pivotal role in a unique post-transcriptional level stimulates inducible promoter and introduced into HTC cells. The addition of dexamethanegative feedback system that prevents ODC degradation. sone, a synthetic glucocorticoid, to excess accumulation of cellular polyamines by both accelerating ODC Antizyme recycles to promote ODC stable transfected HZ7 cells, increased the amount of Zl mRNA and induced degradation and suppressing polyamine degradation Antizyme was discovered in 1976 active antizyme in the absence of uptake. This review will briefly summarize how these unique mechanisms by Canellakis and co-workers as a exogenous polyamines. Addition of dexapolyamine-inducible ODC inhibitor in methasone to cells with high levels of rat liver and several lines of cultured pre-induced ODC elicited a rapid decay S. Hayashi,Y. Murakami and S. Matsufuji are cells 12,13. It is a 26.5kDa protein that of ODC after a short lag time, which at the Departmentof BiochemistryII, The can reversibly bind to ODC. The induc- could be prevented with cycloheximide. Jikei UniversitySchoolof Medicine,Minato-ku, tion of antizyine by polyamines is These results were similar to those Tokyo105, Japan.
Ornithine decarboxylase antizyme: a novel type of regulatory protein
Shin-ichi Hayashi, Yasuko Murakami and Senya Matsufuji
9 1996,ElsevierScienceLtd
27
REVIEWS
TIBS 2 1 - JANUARY 1996
ODC dimer
Omithine
Figure 1 Antizyme-mediated negative feedback regulation of ornithine decarboxylase (ODC) and polyamine uptake. Active ODC comprises two identical monomer subunits with two active sites formed at the interface. The enzymatically active dimeric form of ODC is in rapid equilibrium with the inactive monomeric form. Antizyme (AZ), which is induced by translational frameshifting, preferentially binds with an inactive ODC monomer to form an ODC-antizyme complex. This binding causes a conformational change of the ODC subunit, exposing the carboxy-terminal region to attack from the 26S proteasome complex. ODC is broken down by the proteasome to a mixture of peptides of 5-11 amino acid residues, whereas antizyme is released and recycled to destabilize more ODC monomers. Antizyme also inactivates polyamine uptake at the cell membrane, and is bound by an antizyme inhibitor that has a higher affinity for antizyme than ODC does, and apparently stabilizes ODC by reducing the amount of antizyme available to ODC destabilization.
observed upon addition of polyamines, and clearly indicate that antizyme accelerates ODC degradation in whole cells independently of polyamines, and that the polyamine effect to elicit ODC decay is mediated mostly, if not totally, by antizyme ~9. Further evidence was obtained in studies with an in vitro ODC-degradation system. Kahana and co-workers developed a reticulocyte lysate system in which ODC synthesized in vitro was degraded in the presence of ATP2~ In their system, however, exogenously added ODC was not degraded, leaving some doubt as to the authenticity of the system. In cell-free extracts from both Chinese hamster ovary (CHO) cells and HTC cells, exogenously or endogenously labeled ODC is degraded in the presence of ATP and antizym e21. Omission of either ATP or antizyme results in a marked suppression of ODC degradation. The rate of ODC degradation is accelerated by increasing the amount of antizyme to a maximum when the antizyme to ODC ratio is about 1:1. It was also demonstrated that Kahana's system reflects ODC degradation in whole cells; the reticulocyte lysate system contains a very small amount of endogenous antizyme, which is sufficient for degradation of small amounts of endogenous ODC, but not for relatively large amounts of
28
exogenous ODe 22. Li and Coffino observed that decay of ODC synthesized in vitro was stimulated by in vitro-synthesized antizyme in reticulocyte lysates 23.
Involvement of the protease Three major groups of proteases are
known to be present in cells; lysosomal cathepsins 24, Ca2§ cytosolic calpains 25 and proteasomes 2e. Inhibitor studies have led to the suggestion that proteasomes are the most probable candidates to be involved in ODC degradation. In collaboration with Tanaka's group from Tokushima University, we demonstrated that ODC-degrading activity is completely inhibited by treatment with an anti-proteasome antibody27. Two kinds of proteasomes are known to be present in the cytosol, namely the 20S proteasome made up of 15-20 different subunits, and the 26S proteasome, which consists of the 20S proteasome core associated with 15-20 regulatory subunits 2~. In a highly purified fraction of 26S proteasome (isolated on a glycerol density gradient), ODC-degrading activity exhibited a pattern identical to the proteolytic activities of the 26S proteasome 27, which was of particular interest because this proteasome was thought to degrade poly-ubiquitinated proteins only. However, ODC is well degraded in
a reconstituted system consisting of purified 26S proteasome, antizyme and ATP, but lacking ubiquitin or related enzymes. This is the first case of a nonubiquitinated protein being degraded by the 26S proteasome, and the first example of the in vitro degradation of a cellular protein by a specific ATPdependent protease. ATP is required for both ODC degradation and for the association of the 20S proteasome with regulatory subunits to form the 26S proteasome. Here, ATP is required as an energy source since non-metabolizable ATP analogs cannot be used instead 27. However, the mechanism for this ATP requirement remains to be clarified.
Structural considerationsof ODC Mouse ODC consists of two identical subunits of 461 amino acids. Two active sites formed with amino acids from both subunits are present at the interface. The active ODC homodimer is in equilibrium with the inactive monomer, to which antizyme preferentially binds 2s (see Fig. 1). Structural elements of ODC responsible for its degradation have been pursued by Coffino and co-workers who used various deletion mutations and chimeric variants of ODC. The carboxy-terminal region of ODC (amino acids 423-461), which is missing in stable trypanosomal ODC, is essential for both antizyme-dependent and
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T I B S 2 1 - JANUARY 1 9 9 6
constitutive degradations of ODe 23'29. The region contains one of two PEST regions in mouse ODC (amino acids 423--449). PEST regions are rich in proline (P), glutamic acid (E), serine (S) and threonine (T), and are thought to be a signal for selective proteotysis (PEST hypothesis3~ In addition, a single amino acid exchange in this carboxyterminal region (Cys441 to Try441) results in stable ODC in HMOA cells 31. Furthermore, a region of mouse ODC (amino acids 117-140) is needed for binding with antizyme, whereas another internal PEST region (amino acids 298--333) appears not to be associated with ODC instability.
Antizyme functional domain Coffino's group have shown that the carboxy-terminal half of antizyme (amino acids 106-212) alone can bind with ODC and inactivate it, but an adjacent region (amino acids 55-105) is necessary for destabilizing ODC33. Our own experiments agreed with this result32. These observations suggest that the binding of antizyme to an ODC subunit not only prevents reassociation of inactive subunits, but also causes a conformational change of the subunit. This results in exposure of the ODC carboxy-terminal region to attack from the 26S proteasome. Most antizyme molecules are then liberated intact and recycled to destabilize another ODC subunit (Fig. 1). Chromatographic analysis of the ODC degradation products showed that it is broken down into many different
peptides of 5-11 amino acids. However, free amino acids were not detected, indicating that ODC is degraded by endoproteolysis34. This could have implications for the participation of the 26S proteasome in the processing of class I major histocompatibility complex (MHC) antigenic peptides.
Polyamine-stimulated translational frameshifting As described earlier, induction of antizyme is not dependent on mRNA synthesis. Northern blot analysis showed that antizyme mRNA is present in substantial amounts in all rat tissues examined, either with or without polyamine treatment35. Consistent with this, the antizyme gene has a potent promoter activity36 and antizyme mRNA has a long half-life (12 hours) 35. When full-length cDNAs and a genomic clone36 of rat antizyme were sequenced, an unusual structure for antizyme mRNA37 was revealed. Close to the 5' end is an open reading frame (ORF1) that is too short to encode antizyme. A long overlapping ORF (ORF2), containing the +1 frame with respect to ORF1, lacks an AUG initiator codon, although this frame was used for the expression of Zl antizyme in transfected HZ7 cells using an artificial initiator. The discontinuity of the reading frames is conserved in both human38 and frog39 antizymes. Therefore, an unusual mechanism involving either translational frameshifting or RNA editing, is needed to translate antizyme mRNA. Ample production of
full-length antizyme was observed when a variant antizyme mRNA, lacking a single nucleotide at the termination codon of ORF1, was translated using a rabbit reticulocyte lysate system. By contrast, there was hardly any translation of wild-type antizyme mRNA in the absence of polyamines. Addition of spermidine stimulated translation in a dose-dependent manner with an optimum effect at 0.6raM. The mRNA sequence recovered from the lysate after translation remained unchanged, thus excluding the possibility that the event is a result of RNA editing. Therefore, these results indicate that polyamines induce antizyme by stimulating programmed translational frameshifting4~ Rom and Cahana obtained similar results with antizyme cDNA4~ and the amino acid sequence at the frameshift site was determined using a reporter system. The UGA termination signal and a pseudoknot structure were also identified as downstream stimulators. Although an increasing number of programmed frameshifts have been found to be involved in the gene expression of eukaryotic viruses, the antizyme gene is the first eukaryotic cellular gene known to require frameshifting for its expression. Also, this is only the second reported case of programmed frame~ shifting used for regulation, the first being bacterial polypeptide chain release factor-2. The detailed molecular mechanism, in particular, how polyamines stimulate the frameshift, remains to be clarified by future studies.
Table I. ODC characteristics in different organisms ODC structure
Turnover rate of ODC
Organism
Identity in antizymebinding region a
Carboxy-terminal region b
PEST~ region
- Polyamine
+ Polyamine
Presence of antizyme
Mouse
24
0
+
Moderate
Rapid
+
Chicken
18
0
+
Moderate
Rapid
+
Xenopus
20
0
+
Moderate
Rapid
+
9
-49
?
ND
ND
ND
Saccharomyces cerevisiae
13
-48
+
Moderate
Moderate
Neurospora crassa
11
-41
+
Slow
Moderate
6
-28
+
Slow
Slow
12
-36
-
Slow
Slow
Drosophila
Leishmania Trypanosoma
ND
aNumber of amino acids identical to antizyme-binding region of mouse ODC, which is composed of 24 amino acids. bNumber of deleted amino acids compared with mouse ODC. CRich in proiine, glutamic acid, serine and threonine. Putative signals for selective proteolysis. ND, not determined.
29
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Antizyme regulates polyamlne levels
and also has a structure that requires 18 Murakami, Y., Matsufuji, S., Nishiyama, M. and Hayashi, S. (1989) Biochem. J. 259, 839-845 frameshifting for translation 4~ Rapid 19 Murakami, Y., Matsufuji, S., Miyazaki, Y. and ODC turnover and antizyme-mediated Hayashi, S. (1992) J. Biol. Chem. 267, polyamines. Mitchell and co-workers feedback regulation also appear to 13138-13141 found that the involvement of an operate in insect cells (S. Hayashi, 20 Bercovich,Z., Rosenberg-Hasson,Y., Ciechanover,A. and Kahana, C. (1989) J. Bio/. uncharacterized short-lived protein unpublished). Among lower eukaryotes, Chem. 264, 15949-15952 induced by polyamines is essential in such as Saccharomyces and Neurospora, 21 Murakami, Y. et aL (1992) Biochem. J. 283, this process. In their DFMO-resistant ODC turns over moderately rapidly and 661-664 variant cell line derived from HTC cells, antizyme is not detected. In parasites, 22 Murakami, Y. eta/. (1993) Biochem../. 295, 305-308 both ODC and polyamine transporP 2 such as Trypanosoma and Leishmania, 23 Li, X. and Coffino, P. (1993) Mo/. Cell. Biol. 13, are stable and neither are subject to ODC is very stable and antizyme is 2377-2383 feedback regulation by polyamines. not present. Although the presence of 24 Saido, T. C., Sofimachi, H. and Suzuki, K. (1994) FASEB./. 8, 814-822 Recently, it was found that the trans- antizyme-like proteins has been reT. et al. (1993) J. Struct. Biol. 111, fected antizyme gene was induced by ported in prokaryotes and plants, of 25 Yoshimura, 200-211 dexamethasone-suppressed polyamine which some have been well character- 26 Ciechanover,A. (1994) Cell 79, 13-21 transport in HZ7 ceils 43. In collabo- ized 46,47,their physiological roles remain 27 Murakami, Y. et al. (1992) Nature 360, 597-599 28 Mitchell, J. L. A. and Chen, H. J. (1990) ration with lgarashi's group from Chiba to be clarified by future studies. Biochim. Biophys. Acta 1037, 115-121 University, we obtained the same 29 Li, X. and Coffino, P. (1992) Mol. Cell. Biol. 12, results ~. Thus, antizyme effectively 3356-3362 References 30 Rechsteiner, M. (1988) Adv. Enz. Regul. 27, suppresses cellular polyamine levels by 1 Tabor,C. W. and Tabor, H. (1984) Annu. Rev. 135-151 both destabilizing ODC and inactivating Biochem. 53, 749-790 31 Miyazaki, Y., Matsufuji, S., Murakami, Y. and 2 Scalabrino, G. (1994) Curr. Trends Exp. the polyamine uptake system (Fig. 1). Hayashi, S. (1993) Eur. J. Biochem. 214, Endocrinol. 2, 153-183 It would be exciting to find that dis837-844 3 Auvinen, M., Paasinen, A., Anderson, A. C. and 32 Li, X. and Coffino, Y. (1994) Mo/. Cell Bio/. 14, ruption of antizyme function is related HOItt~, E. (1992) Nature 360, 355-358 87-92 4 Tamori, A. et al. (1995) Cancer Res. 55, to human disease. As mentioned before, 33 Ichiba, T. et al. (1994) Biochem. Biophys. Res. 3500-3505 ODC is now considered to be a possible Commun. 200, 1721-1727 5 Hayashi, S. (1989) in Ornithine Decarboxylase: proto-oncogene product 3,4 and there34 Tokunaga,F. et al. (1994) J. Biol. Chem. 269, Biology, Enzymolof~y,and Molecular Genetics 17382-17385 fore, antizyme might act as a tumor sup(Hayashi, S., ed.), pp. 35-45, PergamonPress 35 Matsufuji, S. eta/. (1990) J. Biochem. 108, 6 Murakami, Y., Matsufuji, S., Miyazaki, Y. and pressor. This possibility will be tested 365-371 Hayashi, S. (1994) Biochem. J. 304, 183-187 by using chromosomal mapping and by 36 Miyazaki, Y., Matsufuji, S. and Hayashi, S. 7 Hayashi, S. and Canellakis, E. S. (1989) in deactivating the antizyme gene. (1992) Gene 113, 191-197 Omithine Decarboxylase, Biology, Enzymolog)/, Polyamine transport is known to be under negative feedback control by
and Molecular Genetics (Hayashi, S., ed.),
Concludingremarks The regulation of ODC degradation has many unique features, such as the extreme rapidity of ODC degradation, ubiquitin-independent degradation by the 26S proteasome, frameshift-dependent translational regulation and the dual action of antizyme. It would be intriguing to discover how such a unique regulatory system has been developed during biological evolution. A comparative biochemical approach should give us some hints on that question (Table I). So far, the rapid antizymedependent turnover of ODC has been observed in mammals, avians (chicken) and amphibians (frogs). Frog antizyme mRNA has 65% identity to rat mRNA,
pp. 47-58, PergamonPress 8 Hayashi, S. and Murakami, Y. (1995) Biochem. J. 306, 1-10 9 L6vkvist, E., Strejnborg, L. and Persson, L. (1993) Eur. J. Biochem. 215, 753-759 10 Kanamoto, R., Utsunomiya, K., Kameji, T. and Hayashi, S. (1986) Eur. J. Biochem. 154, 539-544 11 H61tt~, E. and Pohjanpelto, P. (1986) J. Biol. Chem. 261, 9502-9508 12 Fong,W. F., Heller, J. S. and Canellakis, E. S. (1976) Biochim. Biophys. Acta 428, 456-465 13 Heller, J. S., Fong, L. S. and Canellakis, E. S. (1976) Proc. Natl Acad. Sci. USA 73, 1358-1362 14 Mitchell, J. L. A., Mahan, D. W., McCann, P. P. and Qasba, P. (1985) Biochim. Biophys. Acta 840, 309-316 15 Murakami, Y., Fujita, K., Kameji, T. and Hayashi, S. (1985) Biochem J. 225, 689-697 16 Murakami, Y. and Hayashi, S. (1985) Biochem. J. 226, 893-896 17 Fujita, K., Murakarni, Y. and Hayashi, S. (1982) Biochem. J. 204, 647-652
37 Matsufuji, S. et aL (1995) Cell 80, 51-60 38 Tewafi, D. S. eta/. (1994) Biochim. Biophys. Acta 1209, 293-295 39 Ichiba, T., Matsufuji, S., Miyazaki, Y. and Hayashi, S. (1995) Biochim. Biophys. Acta
1262, 83-86 40 Gesteland, R. F., Weiss, R. 8. and At~ns, J. F. (1992) Science 257, 1640-1641 41 Rom, E. and Cahana, C. (1994) Proc. Natl Acad. Sci. USA 91, 3959-3963. Correction: Proc. Nat/ Acad. Sci. USA 91, 9195 42 Mitchell, J. L. A., Hoff, J. A. and BareyaI-Leyser,A. (1991) Arch. Biochem. Biophys. 290, 143-152 43 Mitchell, J. L. A., Diveley,R. R. and BareyalLeyser, A. (1992) Biochim. Biophys. Acta 1136,
136-142 44 Mitchell, J. L. A., Judd, G. G., BareyaI-Leyser,A. and Ling, S. Y. (1994) Biochem. J. 299, 19-22 45 Suzuki, T. et al. (1994) Proc. Nat/Acad. Sci. USA 91, 8930--8934 46 Panagiotidis, C. A. and Canellakis, E. S. (1984) J. Biol. Chem. 259, 15025-15027 47 Canellakis, E. S. et al. (1993) Proc. Nat/Acad. ScL USA 90, 7129-7133
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