InteractiveFly: GeneBrief

Ornithine decarboxylase antizyme : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - Ornithine decarboxylase antizyme

Synonyms - gutfeeling (guf)

Cytological map position - 48E4

Function - ornithine decarboxylase antizyme

Keywords - protein degradation, target of sex determination hierarchy, cell cycle

Symbol - Oda

FlyBase ID: FBgn0014184

Genetic map position -

Classification - antizyme

Cellular location - cytoplasmic



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

The sex determination master switch, Sex-lethal, regulates the mitosis of early germ cells in Drosophila. Sex-lethal is an RNA binding protein that regulates splicing and translation of specific targets in the soma, but the germline targets are unknown. In an immunoprecipitation experiment aimed at identifying targets of Sex-lethal in early germ cells, the RNA encoded by gutfeeling, the Drosophila homolog of ornithine decarboxylase (ODC) antizyme (Salzberg, 1996), was isolated (Vied, 2003).

Mammalian Antizyme negatively regulates ODC catalytically as well by directing the inactivated enzyme to the proteasome for degradation. This negative regulation of ODC is part of a feedback loop that controls the levels of polyamines within the cell. Translation of Antizyme is dependent on ribosomal frameshifting, which is promoted by high levels of polyamines. As polyamine levels in the cell rise, more Antizyme is synthesized, leading to the turnover of ODC. Polyamines have been implicated in many processes, including cell growth, transcription, and differentiation (Vied, 2003 and references therein). In mammals Antizyme and ubiquitin are thought to be respectively two types of proteasome targeting devices that mark proteins for both ubiquitin-independent and ubiquitin-dependent degradation by the 26 S proteasome (see Gruendler, 2001).

Drosophila gutfeeling interacts genetically with Sex-lethal. It is not only a target of Sex-lethal, but also appears to regulate the nuclear entry and overall levels of Sex-lethal in early germ cells. This regulation of Sex-lethal by gutfeeling appears to occur downstream of the Hedgehog signal. Gutfeeling appears to regulate the nuclear entry of Cyclin B as well. Hedgehog, Gutfeeling, and Sex-lethal function to regulate Cyclin B, providing a link between Sex-lethal and mitosis (Vied, 2003).

Sxl is necessary for female germ cell development regulating mitosis and the female-specific function of recombination. The targets and mechanism by which Sxl controls these processes are not known. Mitotic germ cells reside in the germarium, which is the anteriormost structure of the ovariole. Each of the 14-16 ovarioles of the ovary contains 2-3 stem cells, which divide to produce a cystoblast and another stem cell. Cystoblasts undergo 4 synchronous mitotic divisions with incomplete cytokinesis to form an interconnected 16-cell cyst. One of these 16 cystocytes becomes the oocyte, and the remaining 15 become nurse cells. This occurs as the 16-cell cyst progresses down the germarium. Toward the posterior of the germarium cells of somatic origin, the follicle cells, surround each cyst. This encapsulation is followed by the cyst pinching off from the germarium to produce an egg chamber. The egg chamber moves down the ovariole as it matures into an egg (Vied, 2003 and references therein).

Given the requirement of Sxl in the germline, attempts were made to identify its RNA targets by immunoprecipitation of the protein. To enrich for early germ cells, where Sxl impacts mitosis, advantage was taken of specific female sterile alleles (the Sxlfs alleles) which arrest ovarian development at this stage. In these cells, Sxl protein is expressed at high levels and localized primarily in the cytoplasm as seen in the stem cells and cystoblasts. The mutant Sxl protein fails to undergo the dramatic downregulation that normally occurs as the germ cells mature. One of the RNA molecules identified in the immunoprecipitations is encoded by the gutfeeling (guf) gene, the Drosophila ornithine decarboxylase (ODC) antizyme (Vied, 2003).

The connection of Guf with Sxl provides a potential link between Sxl and mitosis. It has been demonstrated in vertebrate cells that both ornithine decarboxylase and polyamine levels undergo changes that coincide with cell cycle transitions and that depletion of polyamines results in a cell cycle arrest. The polyamine synthetic pathway has also been shown to regulate Cyclin B mRNA levels in vertebrate cells. When ODC is inhibited, cyclin B mRNA levels increase. More directly, Guf affects the nuclear localization of Cyclin B, as it does of Sxl. In addition, the data show that the nuclear entry of Cyclin B depends on Sxl. Typically, Cyclin B regulates cell cycle transitions, but this does not appear to be the case in germ cells. Unlike Cyclin A, whose levels change with the cell cycle, Cyclin B levels remain constant in early germ cells. Additionally, Cyclin A associates with the fusome during the G2 phase of the cell cycle, while Cyclin B does not. This suggests that Cyclin A and Cyclin B regulate early germ cell mitosis by different mechanisms. Overexpression of Cyclin B (or Cyclin A) results in only one extra round of cystocyte divisions to produce a 32-cell cyst, and it has been proposed that Cyclin B (and Cyclin A) regulate specialized cystocyte divisions. Cyclin B is downregulated at approximately the 2-cell cyst stage (the same as Sxl) and is not detected again until the 16-cell stage. This timing may reflect a specific requirement for Cyclin B in one of the 4 mitoses that generate the 16-cell cyst. The dependence of Cyclin B nuclear entry on Sxl suggests how Sxl might regulate germ cell mitosis (Vied, 2003).

The genetic data suggest that the effect of Guf on Sxl and Cyclin B is downstream of the Hh signal. In early germ cells, Sxl is in a complex with the cytoplasmic components of the Hh signaling pathway. While the data do not provide evidence to the point of action, it is suggestive that Guf promotes the disassembly of this complex with Sxl. Given that Antizyme (Guf) binds directly to the vertebrate ODC and Smad1 proteins and targets them to the proteasome (reviewed by Coffino, 2001a and b and Hoyt, 2003), it is tempting to speculate that the disassembly of the Hh cytoplasmic components involves targeting of one or more of the components to the proteasome. Additionally, it has been demonstrated that the inhibitory cytoplasmic NF-kappaB complex can be dissociated by changes in polyamine levels. Inhibiting ODC and thus depleting polyamine levels, a condition equivalent to Antizyme overexpression, leads to the proteolysis of Ikappa-Balpha. NF-kappaB dissociates from the cytoplasmic complex and enters the nucleus where it affects transcription. These effects are analogous to the role proposed for Guf in dissociating the components of the Hh cytoplasmic complex (Vied, 2003).

In its effects on Sxl, guf appears to act downstream of Hh and upstream of the cytoplasmic components. If guf functions downstream of Hh, it does not appear to do so in all Hh-regulated ovarian processes. This is exemplified by the lack of an effect on the turnover of the fusome. Also, guf does not affect the known role of Hh in regulating follicle cell proliferation. Finally, a connection between Cyclin B and the Hh signaling pathway has been previously described, but in a different context. Patched 1 (Ptc 1), which is one of two Ptc proteins in mammals, has been shown to directly interact with Cyclin B, inhibiting its nuclear translocation until cells were exposed to the Hh signal. Whether Ptc directly interacts with Cyclin B in Drosophila remains to be determined. These results, nevertheless, provide an intriguing parallel linking the Hh signal and Guf to the nuclear translocation of Sxl and Cyclin B (Vied, 2003).


GENE STRUCTURE

The Drosophila guf locus appears to have four promoters because four different cDNAs are generated. Each cDNA has a different 5' exon with the translation start site (except for the second 5' exon, which does not contain a start codon) which is spliced to two common exons, the first of which contains the translation frameshifting site (Ivanov, 1998). Interestingly, the polypyrimidine tract at the 3' splice site of the first common exon has a run of eight Uridines (U). This is a feature found in all established Sxl targets: msl-2, tra, and Sxl. Additionally, guf has several long polyU tracts in the introns of all its transcripts. For splicing regulation in vivo, Sxl shows a requirement of at least seven Us at its binding site, while runs of six Us gives compromised regulation. In vitro, shortening of target polyU tracts below six severely decreases the affinity of Sxl protein for its RNA (Vied, 2003).

In the 3' UTR short, six nucleotide-long, polyU runs exist in the mature guf message. This raised the question of whether Sxl regulates guf primarily through splicing or translation control. To address this, Sxl was immunoprecipitated from extracts of both wild-type and Sxlf4 ovaries, and the RNA in the immunoprecipitates was examined by RT-PCR with guf-specific primers. The primers span the two introns of the smallest guf transcript, so the size of the PCR products allows for distinguishing between spliced and unspliced forms of guf RNA (Vied, 2003).

A Drosophila antizyme with high homology to the sequence for mammalian antizyme (ornithine decarboxylase antizyme) has been reported. Homology of this coding sequence to its mammalian antizyme counterpart also extends to a 5' open reading frame (ORF) which encodes the amino-terminal part of antizyme and overlaps the +1 frame (ORF2) that encodes the carboxy-terminal three-quarters of the protein. Ribosomes shift frame from the 5' ORF to ORF2 with an efficiency regulated by polyamines. At least in mammals, this is part of an autoregulatory circuit. The shift site and 23 of 25 of the flanking nucleotides (which are likely important for efficient frameshifting) are identical to their mammalian homologs. In the reverse orientation, within one of the introns of the Drosophila antizyme gene, the gene for snRNP Sm D3 is located. Previously, it was shown that two closely linked P-element transposon insertions caused the gutfeeling phenotype of embryonic lethality and aberrant neuronal and muscle cell differentiation. The present work shows that defects in either snRNP Sm D3 or antizyme, or both, are likely causes of the phenotype (Ivanov, 1998).

Programmed frameshifting is known to occur in single-celled eukaryotes (yeast and protozoa) and in Xenopus but has not previously been discovered in any intermediate organism. The finding of antizyme programmed frameshifting in Drosophila provides the opportunity to study the evolution of this recoding event, which involves a transitory alteration of the rules of readout of the genetic code. The pseudoknot, a 3' mRNA message feature that is an important stimulator for the recoding signal in mammalian systems, is not recognizable in Drosophila, and the identification of its presumed alternative is of major interest. Of the three known RNA elements that stimulate antizyme frameshifting in mammalian cells, the other two are present in guf. One of these is the UGA stop codon of ORF1. It is interesting that even though in vitro translation experiments have shown that the other two stop codons (UAG and UAA) are almost as effective in stimulating frameshifting in decoding mammalian antizyme sequences so far all eukaryotic antizyme genes have UGA as the stop codon of ORF1. Another stimulatory element, a sequence immediately 5' of the UGA stop codon, is also likely to be present in the D. melanogaster antizyme sequence. Of 18 nucleotides 5' of the UGA stop codon, 16 are identical for guf and the rat antizyme sequence (with one of the two mismatches being an antizyme polymorphism). This level of 5' sequence homology is striking and provides a clear indication that this region plays an important role in antizyme frameshifting. The apparent absence of an RNA pseudoknot 3' of the UGA stop codon of guf1 is puzzling. The nucleotides within the stems of this pseudoknot are absolutely conserved among all known vertebrate antizyme sequences. Despite the apparent absence of a 3' pseudoknot, there is nucleotide sequence homology between this region of guf and vertebrate antizyme sequences. Perhaps some other RNA structure is present in this region of guf. Indirect evidence supports such a hypothesis. Comparison of D. melanogaster and D. virilis antizyme nucleotide sequences reveals that the 53 nt between the UGA of the putative frameshift site and intron 2 (a region most likely to contain a stimulatory 3' RNA structure) are completely conserved between the two species, even though the conservation of the rest of the known exonic sequences is only 75%. Computer programs predict several stem loops in this region, but the relevance of any of them to translational frameshifting is unknown. Even though additional frameshift-stimulatory elements in the Drosophila antizyme sequence are not obvious, there is a strong indication that there is more to the guf frameshift site than the 28-nt region homologous to the mammalian antizyme frameshift site. The 28 nt alone cannot stimulate more than 3% frameshifting in vitro. The data for the in vitro translation of guf strongly suggests that the frameshift efficiency in that system is much higher than 3%, thus implying the existence of additional frameshift-stimulatory signals in Drosophila antizyme mRNA (Ivanov, 1998).


REGULATION

Splicing of guf mRNA

Both forms of guf RNA are readily detected although the yield of mature RNA is greater than the precursor transcript in both wild-type and Sxlf4 ovaries. The overall yield from wild-type ovaries was lower for both products, but since the immunoprecipitations and RT-PCRs are not quantitative, this comparison is only speculative. To determine whether Sxl also binds guf RNA in somatic tissues, the immunoprecipitation and RT-PCR performed using embryonic extracts. Sxl also binds to spliced and unspliced guf RNA in embryos (ratio of spliced to unspliced is approximately equal). These data suggest that Sxl may regulate both the splicing and translation of guf (Vied, 2003).

Since Sxl binds to guf RNA in both the soma and germline, RT-PCR amplifications were performed for the four guf transcripts to determine whether any sex-specific products could be detected. guf RNA was analyzed from males and females (whole animals and carcasses) as well as testes and ovaries. For all cDNA types, no significant difference between the sexes was observed. From these data, it is concluded that Sxl does not regulate the alternative splicing of guf RNA in a global manner. Given the general requirement for guf, splicing regulation of guf RNA by Sxl may be restricted to only a subset of cells, such as the early germ cells, making detection of altered transcripts unlikely. Since there are no apparent alternative exons within guf, it is also possible that Sxl causes a retention of the introns leading to the degradation of the RNA. Alternatively, the regulation by Sxl may occur primarily at the level of translation control (Vied, 2003).

While the RNA binding data suggest that guf is a target of Sxl, a genetic interaction between the two genes would strengthen the idea that the two genes have a related function. Therefore the dose of guf was reduced in homozygous Sxlf4 females, which normally have small ovaries of tumorous egg chambers. This was done by using a P-element insertion allele (guf118-3) or a small deletion allele produced by the imprecise excision of the guf118-3 P-element (guflex47; Salzberg, 1996). Reducing guf dose in a homozygous Sxlf4 background rescues the Sxlf4 phenotype. The females lay eggs that hatch and produce adults (Vied, 2003).

Since the original description of guf, an additional gene, SmD3, was found in the intron of guf (Ivanov, 1998). SmD3 encodes a snRNP (small nuclear ribonucleoprotein) common to splicing snRNPs and is transcribed in the opposite direction to guf. More relevant to the existing guf alleles is that the P-element insertions that uncover guf are likely to be affecting both genes. The P-elements are inserted in the 5' UTR of SmD3, and guf deletion alleles derived from their imprecise excision affect the coding sequences of both genes. Additionally, it was recently suggested that the reported phenotype of guf (Salzberg, 1996), as caused by transposon insertions, is primarily due to the alteration of SmD3 (Schenkel, 2002). Since Sxl is a splicing regulator, it was unclear whether the effects on Sxlfs females were a result of reducing guf or SmD3 or both. Additional experments indicate that the rescue is a result of an interaction of Sxl with guf and not with SmD3. Data also indicate that, in the germline, the guf118-3 allele does affect guf expression. Since reducing the dose of guf allows differentiation of Sxl mutant germ cells, the data suggest that Sxl normally functions as a negative regulator of guf. Presumably, the mutant Sxl proteins are unable to properly regulate the splicing and/or translation of guf RNA (Vied, 2003).


DEVELOPMENTAL BIOLOGY

Embryonic

Northern analysis has revealed that the guf gene is expressed in all stages of embryonic and larval development as well as in pupae and adults. An abundant 2.1-kb transcript is detected in all developmental stages. An additional transcript (-1.6 kb) that labels faintly with guf probes may correspond to a different splicing variant of guf. However, this band could not be detected in other RNA samples prepared from similar developmental stages, suggesting that it may be a degradation product of the 2.1-kb transcript (Salzberg, 1996).

The expression pattern of the guf gene was determined with guf antisense RNA probes using in situ hybridization to whole mount embryos. Low levels of uniformly distributed guf RNA are detected in precellularized embryos, suggesting a maternal contribution of guf transcripts. During gastrulation and germband extension guf transcripts are mostly abundant in the mesoderm and the invaginating posterior midgut. At stage 11, patches of mesodermal cells that, based on their position, probably correspond to fat body precursors begin to express higher levels of guf RNA. A strong signal is also detected at this stage in the Malpighian tubule rudiments and the foregut. As germband retraction takes place, guf expression is maintained in the developing fat body, Malpighian tubules and the posterior and anterior midgut. In addition, at late stage 12 and stages 13-14 guf is expressed at low levels throughout most of the embryo including the subectodermal layer that contains most of the PNS cells. At stage 14, guf transcripts are first detected in the developing body wall muscles. Expression of guf in body wall muscles, fat body and the midgut is maintained throughout the rest of embryonic development (Salzberg, 1996).

The P-element enhancer detectors inserted in the guf gene confer ß-gal expression in a pattern similar to the distribution of guf mRNA. Very high levels of ß-gal activity were detected in the midgut (Kania, 1995). (Due to the strong expression in the gut and the defects in the sensory nervous system caused by these insertions the gene was named gutfeeling). Very weak lacZ expression was detected in the developing somatic muscles starting at stage 14 of embryonic development. Embryos heterozygous for these insertions display weak ß-gal expression in developing muscles, whereas expression in other tissues mentioned above is undetectable (Salzberg, 1996).

Since guf is expressed in Malpighian tubules and fat bodies, the Malpighian tubules and fat bodies of gut minus embryos were examined to determine whether they exhibit morphological defects. Malpighian tubules were visualized by anti-Cut staining and found to be morphologically normal in mutant embryos. Fat bodies were visualized with antibodies against Neurotactin and no obvious morphological defects were observed (Salzberg, 1996).

To assess the extent of maternal contribution of guf mRNA and to determine whether zygotic guf expression is eliminated in a deletion allele, mutant embryos were examined by in situ hybridization with guf antisense RNA probe. This probe did not contain sequences from the 5' terminus that were not removed by this deletion (the probe corresponds to nucleotides 1310-2164 of the cDNA). These experiments revealed that guf mRNA is present in guf homozygous mutant embryos until cellular blastoderm at levels similar to those found in wild-type embryos. The store of maternal guf mRNA is depleted rapidly during gastrulation, and by the end of stage 7 only low levels of residual guf transcripts are detected in the invaginating posterior midgut. No guf expression was detected in guf embryos at later stages of embryogenesis. These data suggest that zygotic guf expression is eliminated in guf mutant embryos (Salzberg, 1996).

If Sxl is a negative regulator of guf, it was reasoned that a correlation might be detectable between the expression of guf RNA and Sxl protein. RNA in situ hybridizations using guf sequences as a probe on wild-type ovaries has shown that the expression of guf is dynamic and changes considerably as the germ cells mature. Early germ cells have very low levels of guf RNA, but further down the germarium, the levels increase. guf RNA is also detected in the follicle cells of the germarium. In stage 2 egg chambers (the first chamber past the germarium), expression decreases to very low levels in both the follicle and germ cells. After stage 2, guf RNA is expressed in the germ cells but is essentially undetectable in the follicle cells. As vitellogenesis begins, the levels in germ cells drop again and guf RNA is detected primarily in the oocyte. After this stage, high levels are seen in the nurse cells and oocyte, while follicle cell expression remains very low (Vied, 2003).

The cells in the germarium that show very low levels of guf RNA are the early germ cells, which also express high levels of cytoplasmic Sxl. guf RNA levels begin to increase in the region of the germarium where Sxl is downregulated, suggesting that Sxl might act negatively on guf. By contrast, in Sxlf4 ovaries, guf RNA levels are found to be high throughout, including the germ cells immediately below the terminal filament. An increase in guf RNA is also seen in Sxlf5 ovaries. These observations support the hypothesis that, normally, Sxl downregulates guf expression and suggest that the mutant Sxlfs proteins are inefficient at this function. Consistent with the observation that decreasing the dose of guf partially rescues the Sxlfs female sterile phenotype, wild-type levels of guf RNA are seen in the germ cells of Sxlf5/Sxlf1;guf118-3/+ ovaries (Vied, 2003).


EFFECTS OF MUTATION

The gutfeeling (guf) gene was uncovered in a genetic screen for genes that are required for proper development of the embryonic peripheral nervous system. Mutations in guf cause defects in growth cone guidance and fasciculation and loss of expression of several neuronal markers in the embryonic peripheral and central nervous systems. guf is required for terminal differentiation of neuronal cells. Mutations in guf also affect the development of muscles in the embryo. In the absence of guf activity, myoblasts are formed properly, but myoblast fusion and further differentiation of muscle fibers is severely impaired. The guf gene was cloned and found to encode a 21-kD protein with a significant sequence similarity to the mammalian ornithine decarboxylase antizyme (OAZ). In mammals, OAZ plays a key regulatory role in the polyamine biosynthetic pathway through its binding to, and inhibition of, ornithine decarboxylase (ODC), the first enzyme in the pathway. The elaborate regulation of ODC activity in mammals still lacks a defined developmental role and little is known about the involvement of polyamines in cellular differentiation. Guf is the first antizyme-like protein identified in invertebrates (Salzberg, 1996).

Mutations in the gutfeeling, gene were uncovered in a genetic screen designed to identify genes that play a role in the development of the embryonic PNS (Kania, 1995). Embryos from 2000 strains carrying homozygous lethal P-element insertions on the second chromosome were stained with monoclonal antibody (MAb) 22C10 and examined for defects in the PNS. The guf gene was identified by three insertional mutations that cause similar phenotypes and were mapped to cytological position 48E5-12. Precise or near precise excision of a P-element insertion in guf reverts both the lethality and the PNS phenotype, demonstrating that the insertion causes the phenotype. Additional guf alleles were generated by imprecise excision of a P-element insertion. One allele corresponds to a small deficiency that removes most of the guf transcription unit and the entire open reading frame (Salzberg, 1996).

This deletion was found to abolish zygotic expression of guf and hereafter is referred to as a null allele. The insertional alleles cause phenotypes that are very similar to those observed in guf null embryos, suggesting that they are severe loss of function or null alleles (Salzberg, 1996).

Mutations in guf are embryonic lethal and cause multiple defects in the embryonic PNS. A smaller number of PNS neurons express 22C10 in homozygous guf embryos than in wild-type embryos and the overall intensity of 22C10 immunoreactivity is much decreased when compared to wild-type embryos (Kania, 1995). The lateral and ventral PNS clusters are more severely affected than the dorsal and ventral clusters. On average, six to seven neurons are labeled with MAb 22C10 in lateral clusters of guf embryos as compared to 10-11 in wild-type, and only three to four neurons label in the ventral cluster of mutant embryos as compared to seven in wild-type. The lateral chordotonal neurons seem to be most severely affected by mutations in guf and only one to three chordotonal neurons are evident in each lateral cluster as compared to five in wild-type embryos. However, the loss of 22C10 immunoreactivity is not restricted to chordotonal neurons and is evident in all types of sensory neurons (Salzberg, 1996).

In addition to the lack of 22C10 expression, PNS axons of guf embryos display severe defects in growth cone guidance and fasciculation. One of the most noticeable defects that is observed in all guf embryos is splitting of the intersegmental nerve (ISN) into two fascicles. The ISN 'loop' phenotype was observed in 57% of abdominal segments in guf homozygous mutant embryos. Crossing of the ISN into neighboring segments is much less frequent and was observed in single segments of -40% of the examined guf null embryos. Defects in growth cone guidance and fasciculation are not restricted to ISNs but are also evident in segmental nerves (Salzberg, 1996).

MAb 22C10 is a relatively late marker for PNS development, since neuronal cells express the 22C10 antigen only after they exit the cell cycle and start differentiating as neurons. Thus, lack of 22C10 expression may indicate a true loss of neurons stemming from a failure of precursor formation, failure of precursor division, transformation of neurons into lineage-related support cells, or cell death. Alternatively, this phenotype may reflect a failure of neuronal cells to fully differentiate and express late neuronal markers. To distinguish among these possibilities, a variety of cell-type-specific markers were used to assess number and identity of PNS cells in guf embryos of various developmental stages (Salzberg, 1996).

Since chordotonal neurons are among the most severely affected neurons the analysis focused on the formation of the conspicuous cluster of lateral chordotonal organs. To determine whether the precursors of chordotonal organs were affected in guf embryos, they were immunocytochemically stained with antibodies against the Atonal protein. The pattern of chordotonal SOPs in the mutant was found to be indistinguishable from that of wild-type embryos. To determine whether SOP division is impaired, the PNS of mutant embryos was examined with anti-Couch potato (Cpo) antibodies. The Cpo protein is present in nuclei of all the cells that constitute each sensory organ. Thus, the number of CPO positive cells reflects the number of cell divisions each precursor went through. The level of CPO protein is reduced in guf mutant embryos when compared to wild-type, but a wild-type number of CPO positive nuclei is evident in each chordotonal organ (Salzberg, 1996).

These observations indicate that zygotic guf is not required for the determination of SOPs or their division but is required during later stages of PNS development. Loss of 22C10 immunoreactivity may reflect a transformation of chordotonal neurons into another type of neurons that normally express lower levels of this antigen. Hence, embryos were stained with anti-Cut antibody that labels only external sensory organs and a subset of multiple dendritic neurons appraisal of whether the identity of chordotonal neurons is altered in guf mutants. The pattern of Cut expression appears normal in guf embryos, indicating no change in the identity of sensory organs. To determine whether the identity of cells within each chordotonal organ is altered, embryos were stained with anti-Prospero to reveal sheath cells, and the anti-Repo/RK2 antibody to reveal ligament cells. Normal numbers of sheath and ligament cells are evident in most lateral chordotonal organs of guf embryos. The results of these experiments suggest that loss of guf activity does not alter the identity of sensory organs, nor does it alter the identity of cells within each organ. It is concluded that guf is not required for cell fate determination but rather for terminal differentiation of the developing PNS cells (Salzberg, 1996).

Whereas cell type-specific antigens are expressed in a normal spatial pattern in the PNS of guf mutants, the level of many markers is greatly reduced. Reduced expression levels were observed for the 22C10 antigen and CPO protein in mature embryos. In addition, the level of the glial homeo protein Repo in ligament cells of the lateral chordotonal organs, but not in exit and peripheral glia, is severely decreased when compared to wild-type embryos. However, the levels of Prospero and Cut proteins in the PNS seem to remain unaffected by mutations in guf. These observations indicate that mutations in guf may exert their effect on late PNS differentiation by altering protein levels (Salzberg, 1996).

Since mutations in guf seem to affect most or all types of sensory neurons, the CNS of mutant embryos was examined to determine whether CNS neurons are affected in a similar fashion. guf embryos exhibited a severe reduction in the number of 22C10-expressing cells in the ventral nerve cord when compared to wild- type embryos. However, the number of neuroblasts in the CNS of stage 10-11 guf embryos appeared normal when visualized with antibodies against Prospero. The overall width of the ventral nerve cord appeared normal when examined with Nomarski optics. This suggests that as in the PNS, the lack of 22C10 expression in the CNS does not reflect a true loss of neuronal cells but a failure of these cells to fully differentiate as neurons (Salzberg, 1996).

To further assess late neuronal differentiation in the CNS, embryos were stained with antibodies against the synaptic vesicle protein Synaptotagmin. This protein serves as a terminal differentiation marker, as expression and synaptic localization of Synaptotagmin just precedes synaptic activity. Synaptotagmin is barely detectable in guf embryos when compared to wild type. This observation again supports the view that mutations in guf impair late neuronal differentiation by altering protein levels (Salzberg, 1996).

To examine the axonal pathways in the CNS of guf mutants, embryos were immunohistochemically stained with MAb 1D4 that recognizes the Fasciclin-II (FasII) protein. In wild-type embryos, anti-FASII staining reveals three distinct longitudinal tracts on either side of the midline. The organization of these tracts in guf embryos is aberrant. Although three distinct fascicles can be identified in some segments, most often only two fascicles are evident and these fascicles appear less tight than normal. The most medial fascicle is the least severely affected, whereas the most lateral fascicle is the most severely affected. In addition, the posterior commissure appears thicker than normal (Salzberg, 1996).

In summary, the defects observed in the CNS of guf embryos are very similar to those observed in the PNS and consist of alterations in the protein levels of neuronal-specific markers coupled to misrouting of axons and defects in fasciculation. These data suggest that guf plays a similar role in PNS and CNS development. However, further analysis of cell identities in the CNS of guf mutants is required to exclude the possible involvement of guf in cell fate determination in the CNS (Salzberg, 1996).

Examination of guf embryos with Nomarski optics indicates that mutant embryos also display defects in muscle development. To study the fully developed muscle pattern, embryos were generated with antibody against Drosophila muscle Myosin. This antibody labels all somatic muscle fibers, the cardial cells and the visceral muscles. Somatic muscles of the body wall are organized in a highly stereotyped pattern in wild-type embryos. This pattern is considerably disrupted in guf homozygous mutant embryos where numerous muscles are missing, displaced or exhibit aberrant shapes (Salzberg, 1996).

In addition, these embryos contain many mononucleate myoblasts that failed to fuse into muscle syncytia. Similar defects were observed in guf mutant embryos stained with antibodies against the spectrin-like MSP-300 protein that is expressed in muscles and chordotonal neurons. The level of MSP-300 protein is greatly reduced in muscle fibers and chordotonal neurons of mutant embryos when compared to wild type (Salzberg, 1996).

Anti-Myosin staining also revealed defects in the visceral and cardiac musculature. In wild-type embryos, a thin layer of evenly spread visceral muscles is detected around the gut. In guf embryos the visceral muscles seem to spread properly around the developing gut, however the thickness of this muscle layer appears less uniform than that of wild-type embryos leading to its rippled appearance. The constrictions of the gut form properly in guf mutants. The contractile part of the heart normally consists of a double row of Myosin expressing cardial cells that form a linear tube underneath the dorsal midline. Although the cardial cells form properly and the assembly of the heart into a linear tube appears relatively normal in guf mutants, Myosin expression in the cardial cells is essentially absent (Salzberg, 1996).

To determine whether the defects observed in the somatic musculature of guf embryos stem from aberrant specification of these muscles or from later differentiation defects, guf embryos were stained for nautilus RNA and for the Evenskipped (Eve) protein. nautilus is normally expressed in a subset of developing muscles in stage 12/13 embryos. This expression pattern does not appear to be altered in guf embryos. The Eve protein is also present in the nuclei of the dorsal-most muscle and pairs of pericardial cells in each abdominal segment. In guf mutants, the number of Eve positive cells appears similar to wild type, but the distribution of nuclei in the dorsal somatic muscle is abnormal. Instead of segregating into two crescent rows of nuclei on either side of the segment, these nuclei remain clumped together. Similarly, these dorsal muscles display abnormal morphology when visualized with anti-Myosin antibody and do not appear as broad and spread out as in wild type. The aberrant distribution of nuclei within muscle fibers is not restricted to the dorsal-most muscles and is evident in all muscle fibers when stained with antibodies against Mef2. These results indicate that the initial specification of individual muscles occurs properly in the absence of guf activity, but myoblast fusion and distribution of the syncytial nuclei within the developing muscle fibers is aberrant. Hence, it has been concluded that in the myogenic lineages, as in the PNS, guf is not required for cell type specification but for terminal differentiation of muscle cells. It is proposed that mutations in guf affect muscle development by reducing the level of various muscle-specific proteins (Salzberg, 1996).

These data suggest that guf plays a role in terminal differentiation of PNS cells following their specification as neurons or support cells but is not required for the specification of cell identities. Based on the observation that the levels of several neuronal-specific proteins are abnormally low in mutant embryos, it is proposed that guf is required for the regulation or maintenance of gene expression in the differentiating PNS. The failure of PNS cells to express late neuronal markers may explain the abnormal morphology of neuronal cells and the defects in growth cone guidance and fasciculation (Salzberg, 1996).

The pattern of body wall muscles is also disrupted in guf mutant embryos since many muscle fibers are missing or misplaced. In addition, the layer of visceral muscles surrounding the gut appears rippled and less regular than normal. Early subdivision of the mesoderm to somatic, visceral and cardiac mesoderm was found to occur normally in the absence of guf, and the initial specification of individual muscles does not seem to be affected. However, myoblast fusion and segregation of the syncytial nuclei within the developing muscles is aberrant leading to abnormal morphology or complete loss of specific muscle fibers. Based on these data it has been proposed that guf is required for differentiation but not cell fate determination of all types of embryonic muscles (Salzberg, 1996).

guf function in female germ cell development

To determine whether guf has a function in female germ cell development, germ cell clones were generated that were homozygous mutant for guf. Clones were induced by using the FLP/FRT mitotic recombination system in a background that had the SmD3+ genomic transgene, to preclude effects of this gene. When induced during the larval stage, clones of germ cells mutant for guf were not recovered in adult ovaries, suggesting a requirement for guf in the early stages of germ cell development. When induced in adults, germ cell clones did not develop beyond stage 2 egg chambers. Within 7 days of induction, many guf mutant cysts were observed in the germarium, and these showed no obvious defects, including their expression of Sxl. However, 14 days after induction, no guf mutant cysts were detected in germaria, but multiple degenerating egg chambers that had been pinched off were observed. These data suggest that, in the adult ovary, guf is not required in the early stages of germ cell development but in the cysts past the germarium, and that guf is needed either for the survival or the prevention of differentiation of germline stem cells. Since guf mutant germ cells show near normal Sxl expression, the data indicate that loss of guf does not have a major effect on Sxl expression in the early dividing germ cells. The proposal that Sxl maintains guf at low levels in region 1 of the germarium is consistent with this view (Vied, 2003).

Germ cells mutant for guf appear to develop through the germarium normally. Therefore whether ectopic expression of guf would affect early germ cell development was examined. Overexpression of Guf causes a decrease in the levels of cytoplasmic Sxl, resulting in fewer germ cells with high levels of the protein under the terminal filament. This effect requires efficient expression of Guf since it is only observed when the frameshift-independent variant of guf, is used. This latter observation also confirms that Guf translation requires frameshifting in vivo (Vied, 2003).

As a different means of increasing Guf levels, wild-type ovaries were treated with polyamines (spermidine). Polyamines increase the translation of endogenous guf RNA by increasing the rate of translational frameshifting. Spermidine causes a decrease in the number of early germ cells with high levels of cytoplasmic Sxl. This effect was observed within 3 h, implying that the change in Sxl is not caused by differentiation of the germ cells. The possibility that spermidine affects Sxl by a Guf-independent mechanism cannot be excluded. However, it is likely that an increase in Guf is involved given the overexpression data above (Vied, 2003).

The effect of Guf overexpression on Sxl in early germ cells strongly resembles the effect of overexpressing the patterning gene, hh (Vied, 2001). In the ovary, Hh is expressed in the terminal filament and cap cells, the somatic cells at the tip of the germarium. Hh regulates the proliferation of the follicle cells of the ovary and has minor effects on germ cell development (Vied, 2003).

To test for an epistatic interaction between hh and guf, hs-hh; guf118-3/+ flies were generated. Removing one copy of guf counteracts the overexpression of Hh, and there is an increase in the number of early germ cells with high levels of cytoplasmic Sxl. Previous data (Vied, 2001) suggested that Hh regulates not only the levels but also the nuclear localization of Sxl in early germ cells. The coding sequence of Sxl suggests a leucine-rich Nuclear Export Signal (NES). Whether the Sxl NES is functional was tested using leptomycin B (LMB). LMB prevents the nuclear export of proteins by binding to exportin 1/CRM1, which binds directly to leucine-rich NES sequences. Wild-type ovaries treated with LMB show an increase in detectable nuclear Sxl. By contrast, early germ cells that have not been treated with LMB show no distinct nuclear foci of Sxl. Consistent with Hh affecting Sxl nuclear entry, overexpression of Hh in the presence of LMB significantly increases the amount of nuclear Sxl. Overexpression of Guf in the presence of LMB has a similar effect, and the effect of Guf is usually stronger than that of Hh (Vied, 2003).

To demonstrate that guf acts downstream of Hh in Sxl nuclear entry, Hh was overexpressed in a background with a reduced dose of guf (hs-hh; guf118-3/+) and the ovaries were treated with LMB. As was seen for the degradation of Sxl, removal of one copy of guf counteracts Hh-regulated nuclear entry of Sxl, and Sxl remained predominantly in the cytoplasm. This observation suggests that Guf functions downstream of Hh in both nuclear entry and degradation, promoting the release of cytoplasmic Sxl in early germ cells (Vied, 2003).

Besides Sxl, Hh affects other germ cell-specific processes. Hh overexpression decreases the ß-Spectrin staining of the fusome. ß-Spectrin is one of the proteins associated with the fusome, a germline specific organelle that plays a role in establishing the interconnected 16-cell cyst. Overexpression of Guf does not appear to affect the fusome. Additionally, reducing the dose of guf does not reverse the effect of Hh overexpression on the fusome. These data implicate guf as a downstream effector of Hh, but only in regulating Sxl (Vied, 2003).

In Drosophila, Cyclin B is not essential for viability but is necessary for female fertility. Since Sxl has been shown to be important for the mitosis of early germ cells, a correlation between Sxl and Cyclin B was sought. In early germ cells, Cyclin B colocalizes with Sxl and is downregulated concomitantly with Sxl. Since Sxl and Cyclin B colocalize in early germ cells, the effect of Hh on Cyclin B was examined. Overexpression of Hh results in fewer Cyclin B-expressing early germ cells, as seen for Sxl. Furthermore, the germ cells that express Cyclin B show reduced levels of the protein. This similarity in response prompted a test to see whether Hh also regulates the nuclear entry of Cyclin B. Overexpression of Hh and treatment with LMB results in higher levels of nuclear Cyclin B than in wild-type early germ cells treated with LMB only. This observation suggests that Hh promotes the nuclear entry of Cyclin B (Vied, 2003).

Since Guf appears to act downstream of Hh to regulate Sxl, whether Cyclin B would also respond to Guf was also examined. Ectopic expression of Guf has the same effect on Cyclin B as on Sxl. Fewer early germ cells with cytoplasmic Cyclin B were observed, and Sxl and Cyclin B continued to colocalize. LMB treatment with Guf overexpression increases the nuclear levels of Cyclin B. As for Sxl, Cyclin B responds more strongly to Guf than Hh (Vied, 2003).

To determine whether Guf also acts downstream of Hh in affecting Cyclin B nuclear entry, hs-hh; guf118-3/+ ovaries treated with LMB were examined. Under these conditions, Cyclin B was predominantly cytoplasmic, suggesting that Guf also functions downstream of Hh in the regulation of Cyclin B (Vied, 2003).

In vertebrates, the intracellular localization of Cyclin B is critical to its function. An NES within the cytoplasmic retention signal (CRS) allows Cyclin B to rapidly shuttle out of the nucleus. Phosphorylation of the CRS results in nuclear accumulation of Cyclin B and its associated Cyclin-dependent kinase with which Cyclin B initiates mitosis. Since Sxl and Cyclin B appear to undergo similar changes in response to Hh and Guf, tests were made to determine whether Cyclin B is affected by changes in Sxl.

Cyclin B was examined in ovaries with mutant Sxl protein. Like Sxl, Cyclin B is localized to the cytoplasm of Sxlf4 germ cells. Treatment of Sxlf4 ovaries with LMB caused the Sxl, but little Cyclin B, protein to accumulate in the germ cell nuclei. As overexpression of Hh with LMB treatment increases the nuclear levels of both Sxl and Cyclin B in wild-type ovaries, the intracellular localization of both proteins was examined in Sxlf4 ovaries after similar treatment. The mutant Sxl protein accumulates in the nuclei, but surprisingly, Cyclin B remains in the cytoplasm. Consistent with this observation, ovaries doubly homozygous for Sxlf4 and Su(fu) treated with LMB show nuclear accumulation of Sxlf4 protein, but not Cyclin B. These results suggest that, normally, Sxl affects the rate of nuclear entry of Cyclin B. Interestingly, the mutations in the Sxlfs alleles alter a region of the protein that is proline-rich. Proline-rich domains have been described as being involved in protein-protein interactions (Vied, 2003).

Sxl can enter the nucleus without the concomitant entry of Cyclin B. Cyclin B mutant ovaries were examined to test the assumption that Sxl does not require Cyclin B for nuclear exiting. A null mutation for cyclin B derived from the imprecise excision of a P-element in the 5' UTR (cycB3) is not lethal except when combined with other cell cycle regulators. The cycB3 mutation results in female sterility with many agametic ovarioles. When germ cells were present, the localization of Sxl was primarily cytoplasmic. Treatment with LMB shows that Sxl is able to enter the nucleus at levels comparable to those observed in wild-type ovaries\. These results suggest that Sxl does not require Cyclin B for nuclear exiting. Otherwise, Sxl would be primarily nuclear in the absence of Cyclin B (Vied, 2003).


EVOLUTIONARY HOMOLOGS

Antizyme function in yeast

The mechanism of the regulatory degradation of ornithine decarboxylase (ODC) by polyamines was studied in fission yeast, Schizosaccharomyces pombe. To regulate cellular spermidine experimentally, S-adenosylmethionine decarboxylase gene (spe2) was cloned and disrupted in S. pombe. The null mutant of spe2 was devoid of spermidine and spermine, accumulated putrescine, and contained a high level of ODC. Addition of spermidine to the culture medium resulted in rapid decrease in the ODC activity caused by the acceleration of ODC degradation, which was dependent on de novo protein synthesis. A fraction of ODC forming an inactive complex concomitantly increased. The accelerated ODC degradation was prevented either by knockout of antizyme gene or by selective inhibitors of proteasome. Thus, unlike budding yeast, mammalian type antizyme-mediated ODC degradation by proteasome is operating in S. pombe (Chattopadhyay, 2001).

The effect of spermidine on the degradation of ornithine decarboxylase has been investigated in S. cerevisiae. In S. cerevisiae, as in other eukaryotic cells, the rate of degradation of ornithine decarboxylase, measured either enzymatically or immunologically, is increased by the addition of spermidine to a yeast culture. It is noteworthy that this effect of added spermidine is found even when the experiments are conducted with strains in which the ornithine decarboxylase is overexpressed several hundred-fold more than the wild-type level. The effect of added spermidine in the overexpressed SPE1 strains is best seen in spe2 mutants in which the initial intracellular spermidine is very low or absent. Experiments with cycloheximide show that new protein synthesis is required to effect the breakdown of the ornithine decarboxylase. These results indicate that S. cerevisiae contains an antizyme-like mechanism for the control of the level of ornithine decarboxylase by spermidine, even though, as contrasted with other eukaryotic cells, no specific antizyme homolog has been detected either in in vitro experiments or in the S. cerevisiae genome (Gupta, 2001).

The expression of mammalian antizyme genes requires a specific +1 translational frameshift
.

Conventional reading of the sequence of ornithine decarboxylase-antizyme mRNA (a protein that modulates the rate of ornithine decarboxylase degradation) results in premature termination at an in-frame termination codon (stop-1), located shortly after the initiation codon. Antizyme expression is demonstrated to require that ribosomes shift from the first open reading frame (termed ORF0) to a second +1 open reading frame (ORF1). These studies show that this frame-shifting, which occurs at maximal efficiency of approximately 20%, is stimulated by polyamines and requires the functional integrity of the stop codon (stop-1) of ORF0. By introducing in-frame deletions, it has been shown that an 87-nt segment surrounding stop-1 enhances frame-shifting efficiency, whereas the 6 nt located just upstream of stop-1 are absolutely essential for this process. Because this segment does not contain sequences that were previously characterized as shifty segments, these results suggest that another mechanism of frame-shifting is involved in mediating antizyme expression (Rom, 1994).

Rat antizyme gene expression requires programmed, ribosomal frameshifting. A novel autoregulatory mechanism enables modulation of frameshifting according to the cellular concentration of polyamines. Antizyme binds to, and destabilizes, ornithine decarboxylase, a key enzyme in polyamine synthesis. Rapid degradation ensues, thus completing a regulatory circuit. In vitro experiments with a fusion construct using reticulocyte lysates demonstrate polyamine-dependent expression with a frameshift efficiency of 19% at the optimal concentration of spermidine. The frameshift is +1 and occurs at the codon just preceding the terminator of the initiating frame. Both the termination codon of the initiating frame and a pseudoknot downstream in the mRNA have a stimulatory effect. The shift site sequence, UCC-UGA-U, is not similar to other known frameshift sites. The mechanism does not seem to involve re-pairing of peptidyl-tRNA in the new frame but rather reading or occlusion of a fourth base (Matsufuji, 1995).

Regulation of ornithine decarboxylase in vertebrates involves a negative feedback mechanism requiring the protein antizyme. A similar mechanism exists in the fission yeast Schizosaccharomyces pombe. The expression of mammalian antizyme genes requires a specific +1 translational frameshift. The efficiency of the frameshift event reflects cellular polyamine levels creating the autoregulatory feedback loop. The yeast antizyme gene and several newly identified antizyme genes from different nematodes also require a ribosomal frameshift event for their expression. Twelve nucleotides around the frameshift site are identical between S. pombe and the mammalian counterparts. The core element for this frameshifting is likely to have been present in the last common ancestor of yeast, nematodes and mammals (Ivanov, 2000a).

Interaction of Ornithine decarboxylase antizyme with Ornithine decarboxylase

Degradation of ornithine decarboxylase, a key enzyme in polyamine biosynthesis, is accelerated by the binding of antizyme, an ornithine decarboxylase inhibitory protein induced by polyamines. The effects of a series of deletion mutants of rat antizyme have been studied. The results indicated that two regions of antizyme, one internal (amino acids 122-144) and the other near the C-terminus (amino acids 211-218) are necessary for its binding to ornithine decarboxylase and inhibition of its activity, and an additional internal region (amino acids 88-118, especially 113-118) is necessary for its destabilization (Ichiba, 1994).

The polyamines spermidine and spermine are ubiquitous and required for cell growth and differentiation in eukaryotes. Ornithine decarboxylase performs the first step in polyamine biosynthesis, the decarboxylation of ornithine to putrescine. Elevated polyamine levels can lead to down-regulation of ODC activity by enhancing the translation of antizyme mRNA, resulting in subsequent binding of antizyme to ODC monomers; this targets ODC for proteolysis by the 26S proteasome. The crystal structure of ornithine decarboxylase from human liver has been determined to 2.1 Å resolution by molecular replacement using truncated mouse ODC. The human ODC model includes several regions that are disordered in the mouse ODC crystal structure, including one of two C-terminal basal degradation elements that have been demonstrated to independently collaborate with antizyme binding to target ODC for degradation by the 26S proteasome. The crystal structure of human ODC suggests that the C terminus, which contains basal degradation elements necessary for antizyme-induced proteolysis, is not buried by the structural core of homodimeric ODC as previously proposed. Analysis of the solvent-accessible surface area, surface electrostatic potential, and the conservation of primary sequence between human ODC and Trypanosoma brucei ODC provides clues to the identity of potential protein-binding-determinants in the putative antizyme binding element in human ODC (Almrud, 2000).

Function of antizyme in protein degradation

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is the most rapidly turned over mammalian enzyme. Its degradation is accelerated by ODC antizyme, an inhibitory protein induced by polyamines. This is a new type of enzyme regulation and may be a model for selective protein degradation. Using a cell-free degradation system, it has been demonstrated that immunodepletion of proteasomes from cell extracts causes almost complete loss of ATP- and antizyme-dependent degradation of ODC. In addition, purified 26S proteasome complex, but not the 20S proteasome, catalyses ODC degradation in the absence of ubiquitin. These results strongly suggest that the 26S proteasome, widely viewed as specific for ubiquitin-conjugated proteins, is the main enzyme responsible for ODC degradation. The 26S proteasome may therefore have a second role in ubiquitin-independent proteolysis (Murakami, 1992).

Regulated degradation of ornithine decarboxylase (ODC) is mediated by its association with the inducible protein antizyme. The N terminus of antizyme (NAZ), although unneeded for the interaction with ODC, must be present to induce degradation. Covalently grafting NAZ to ODC confers lability that normally results from the non-covalent association of native antizyme and ODC. To determine whether NAZ could act similarly as a modular functional domain when grafted to other proteins, it was fused to a region of cyclin B (amino acids 13-90) capable of undergoing degradation or to cyclin B (amino acids 13-59), which is not subject to degradation. The association with NAZ made both NAZ-cyclin B13-90 and NAZ-cyclin B13-59 unstable. Furthermore, NAZ and cyclin B 13-59 are together able to induce in vitro degradation of Trypanosoma brucei ODC, a stable protein. The ODC-antizyme complex binds to the 26 S protease but not the 20 S proteasome, consistent with the observation that ODC degradation is mediated by the 26 S protease. The association is independent of NAZ, suggesting that NAZ does not act as a recognition signal (Li, 1996).

The 26S proteasome is a eukaryotic ATP-dependent protease, but the molecular basis of its energy requirement is largely unknown. Ornithine decarboxylase (ODC) is the only known enzyme to be degraded by the 26S proteasome without ubiquitinylation. The 26S proteasome is responsible for the irreversible inactivation coupled to sequestration of ODC, a process requiring ATP and antizyme (AZ) but not proteolytic activity. Neither the 20S proteasome (catalytic core) nor PA700 (the regulatory complex) by itself contributed to this ODC inactivation. Analysis with a C-terminal mutant ODC revealed that the 26S proteasome recognizes the C-terminal degradation signal of ODC exposed by attachment of AZ, and subsequent ATP-dependent sequestration of ODC in the 26S proteasome causes irreversible inactivation, possibly unfolding, of ODC and dissociation of AZ. These processes may be linked to the translocation of ODC into the 20S proteasomal inner cavity, centralized within the 26S proteasome, for degradation (Murakami, 1999).

The antizyme family consists of closely homologous proteins believed to regulate cellular polyamine pools. Antizyme1, the first described, negatively regulates ornithine decarboxylase, the initial enzyme in the biosynthetic pathway for polyamines. Antizyme1 targets ornithine decarboxylase for degradation and inhibits polyamine transport into cells, thereby diminishing polyamine pools. A polyamine-stimulated ribosomal frameshift is required for decoding antizyme1 mRNA. Recently, additional novel conserved members of the antizyme family have been described. Antizyme2, like antizyme1, binds to ornithine decarboxylase and inhibits polyamine transport. Using a baculovirus expression system in cultured Sf21 insect cells, both antizymes were found to accelerate ornithine decarboxylase degradation. Expression of either antizyme1 or 2 in Sf21 cells also diminished their uptake of the polyamine spermidine. Both forms of antizyme can therefore function as negative regulators of polyamine production and transport. However, in contrast to antizyme1, antizyme2 has negligible ability to stimulate degradation of ornithine decarboxylase in a rabbit reticulocyte lysate (Zhu, 1999).

The bone morphogenetic proteins (BMPs) regulate early embryogenesis and morphogenesis of multiple organs, such as bone, kidney, limbs, and muscle. Smad1 is one of the key signal transducers of BMPs and is responsible for transducing receptor activation signals from the cytoplasm to the nucleus, where Smad1 serves as a transcriptional regulator of various BMP-responsive genes. Based upon the ability of Smad1 to bind multiple proteins involved in proteasome-mediated degradation pathway, whether Smad1 could be a substrate for proteasome was investigated. Smad1 is targeted to proteasome for degradation in response to BMP type I receptor activation. The targeting of Smad1 to proteasome involves not only the receptor activation-induced Smad1 ubiquitination but also the targeting functions of the ornithine decarboxylase antizyme and the proteasome beta subunit HsN3. These studies provide the first evidence for BMP-induced proteasomal targeting and degradation of Smad1 and also reveal new players and novel mechanisms involved in this important aspect of Smad1 regulation and function (Gruendler, 2001).

Smad1 binds to multiple proteins involved in proteasome-mediated degradation pathways such as HsN3, antizyme (Az), and ubiquitin. HsN3 is one of the seven subunits of the 20 S proteasome, the catalytic core of the 26 S proteasome. HsN3 was also shown to be involved in the targeting of p105 NF-kappaB subunit to proteasome for processing. Ubiquitin is well known for its role in covalently modifying proteasomal substrates for ubiquitin-dependent degradation. Az is a protein previously known to bind ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis. Interestingly, its physical interaction with ODC is necessary and sufficient for targeting ODC to the 26 S proteasome for ubiquitin-independent degradation. Thus, ubiquitin and Az are two types of proteasome targeting proteins that mark proteins for both ubiquitin-dependent and ubiquitin-independent degradation by the 26 S proteasome. Currently, it is not clear how proteasome recognizes ubiquitinated proteins or Az-bound ODC. The ability of Smad1 to bind to ubiquitin and Az as well as HsN3, which is a proteasome component, suggests an interesting link between Smad1 and the proteasome targeting events involving ubiquitin, Az and HsN3. Studies were carried out to test whether the physical interaction between Smad1 and proteins involved in proteasomal degradation pathways (HsN3, Az, and Ub) may lead to: (1) proteasomal degradation of Smad1 or (2) proteasomal degradation of Smad1 interacting proteins. Concomitant with these studies, recent studies by others in the field have demonstrated several important roles of proteasomal degradation in regulating the protein levels of Smads and Smad-interacting proteins (28-35). In the signaling pathways of BMPs, it has been shown that Smad1 interacts with an ubiquitin E3 ligase, Smurf1, which regulates proteasomal degradation of Smad1 independent of BMP type I receptor activation. This study provides the first evidence that proteasomal degradation of Smad1 is also induced upon the activation by the BMP type I receptor. Furthermore, the data reveal novel roles of two Smad1 interactors, Az and HsN3, in proteasomal targeting and degradation of Smad1, in addition to Smad1 ubiquitination (Gruendler, 2001 and references therein).

Ornithine decarboxylase (ODC) is regulated by its metabolic products through a feedback loop that employs a second protein, antizyme 1 (AZ1). AZ1 accelerates the degradation of ODC by the proteasome. Purified components were used to study the structural elements required for proteasomal recognition of this ubiquitin-independent substrate. These results demonstrate that AZ1 acts on ODC to enhance the association of ODC with the proteasome, not the rate of its processing. Substrate-linked or free polyubiquitin chains compete for AZ1-stimulated degradation of ODC. ODC-AZ1 is therefore recognized by the same element(s) in the proteasome that mediate recognition of polyubiquitin chains. The 37 C-terminal amino acids of ODC harbor an AZ1-modulated recognition determinant. Within the ODC C terminus, three subsites are functionally distinguishable. The five terminal amino acids (ARINV, residues 457-461) collaborate with residue C441 to constitute one recognition element, and AZ1 collaborates with additional constituents of the ODC C terminus to generate a second recognition element (Zhang, 2003).

The antizymes constitute a conserved gene family with at least three mammalian orthologs. In a degradation system utilizing rabbit reticulocyte lysate, antizyme 1 (AZ1) accelerates proteasomal ornithine decarboxylase (ODC) degradation, but antizyme 2 (AZ2) does not. To examine the relationship between antizyme structure and function, the properties of AZ1 and AZ2 and protein chimeras composed of elements of the two were characterized. AZ1 binds to ODC with about a 3-fold higher potency than AZ2, but this cannot account for their distinct degradative activities. The dissimilar degradative capacity of AZ1 and AZ2 is also observed using purified proteasomes. A series of reciprocal AZ1/AZ2 chimeras was used to determine the sequence elements needed to direct ODC degradation. An element contained within amino acids 130-145 of AZ1 is essential for this function. Constructs in which amino acids 130-145 were exchanged between the antizymes confirmed the critical nature of this region. Within this region, amino acids 131 and 145 proved responsible for the functional difference between the two forms of AZ (Chen, 2003).

Antizyme, polyamines and cell cycle progression

Polyamines are required for entry and progression of the cell cycle. As such, augmentation of polyamine levels is essential for cellular transformation. Polyamines are autoregulated through induction of antizyme, which represses both the rate-limiting polyamine biosynthetic enzyme ornithine decarboxylase and cellular polyamine transport. In the present study it has been demonstrated that agmatine, a metabolite of arginine via arginine decarboxylase (an arginine pathway distinct from that of the classical polyamines), also serves the dual regulatory functions of suppressing polyamine biosynthesis and cellular polyamine uptake through induction of antizyme. The capacity of agmatine to induce antizyme is demonstrated by: (1) an agmatine-dependent translational frameshift of antizyme mRNA to produce a full-length protein and (2) suppression of agmatine-dependent inhibitory activity by either anti-antizyme IgG or antizyme inhibitor. Furthermore, agmatine administration depletes intracellular polyamine levels to suppress cellular proliferation in a transformed cell line. This suppression is reversible with polyamine supplementation. A novel regulatory pathway is proposed in which agmatine acts as an antiproliferative molecule and potential tumor suppressor by restricting the cellular polyamine supply required to support growth (Satriano, 1998).

Cyclical phases of accumulation and depletion of polyamines occur during cell-cycle progression. Regulatory ornithine decarboxylase (ODC) catalyses the first step of polyamine biosynthesis. Ornithine decarboxylase antizyme (OAZ), induced by high polyamine levels, inhibits ODC activity and prevents extracellular polyamine uptake. Spermidine/spermine N1-acetyltransferase (SSAT) regulates the polyamine degradation/excretion pathway. 24 h transient transfection of immortalized human prostatic epithelial cells (PNT1A and PNT2) with antisense ODC RNA or OAZ cDNA, or both, while effectively causing marked decreases of ODC activity and polyamine (especially putrescine) concentrations, results in accumulation of cells in the S phase of the cell cycle. Transfection with SSAT cDNA leads to more pronounced decreases in spermidine and spermine levels and results in accumulation of cells in the G2/M phases. Transfection with all three constructs together produces maximal depletion of all polyamines, accompanied by accumulation of PNT1A cells in the S phase and PNT2 cells in the G0/G1 and G2/M phases. Accumulation of PNT1A cells in the S phase progressively increased at 15, 18 and 24 h of transfection with antisense ODC and/or OAZ cDNA. At 24 h, the DNA content was always reduced, as a possible outcome of altered chromosome condensation. A direct link between polyamine metabolism, cell proliferation and chromatin structure is thus proposed (Scorcioni, 2001).

Antizyme function in spermatogenesis

Studies with mice overproducing ornithine decarboxylase have demonstrated the importance of polyamine homeostasis for normal mammalian spermatogenesis. The present study introduces a likely key player in the maintenance of proper polyamine homeostasis during spermatogenesis. Antizyme 3 is a paralog of mammalian ornithine decarboxylase antizymes. Like its previously described counterparts, antizymes 1 and 2, it inhibits ornithine decarboxylase, which catalyzes the synthesis of putrescine. Earlier work has shown that the coding sequences for antizymes 1 and 2 are in two different, partially overlapping reading frames. Ribosomes translate the first reading frame, and just before the stop codon for that frame, they shift to the second reading frame to synthesize a trans-frame product. The efficiency of this frameshifting depends on polyamine concentration, creating an autoregulatory circuit. Antizyme 3 cDNA has the same arrangement of reading frames and a potential shift site with definite, although limited, homology to its evolutionarily distant antizyme 1 and 2 counterparts. In contrast to antizymes 1 and 2, which are widely expressed throughout the body, antizyme 3 transcription is restricted to testis germ cells. Expression starts early in spermiogenesis and finishes in the late spermatid phase (Ivanov, 2000b).

ODC mRNA and protein are present at some level in all testicular cells (Leydig, Sertoli, spermatogenic cells, etc.), but the levels of expression vary greatly. ODC expression during sperm development increases sharply (from earlier background levels) and peaks in late pachytene spermatocytes and early round spermatids. In later stages of spermatid development and in spermatozoa, ODC expression falls back to background levels. By contrast, no antizyme 3 mRNA expression is detected before early spermatid stages of spermatogenesis or in any of the nonspermatogenic cells of testes. Antizyme 3 mRNA expression is maximal during the middle and late stages (stages VIII through XII) of spermatid development, and then it disappears again in the spermatozoa. It appears that the antizyme 3 wave of expression follows the wave of high ODC expression during spermatogenesis. This pattern of expression of the two genes implies that the physiological role of antizyme 3 is to quickly 'extinguish' (prevent overaccumulation of) ODC activity after the stage at which ODC plays its role in spermatogenesis, most likely late spermatocytic/early spermatidal phase. By the same type of experiments (in situ hybridization), antizyme 1 expression is essentially nondetectable in testicular tissues (specifically during spermatogenesis). This finding is consistent with observations demonstrating that of 50 human tissues examined, antizyme 1 mRNA is least abundant in testis. Because antizyme 2 mRNA was found to be even less abundant in testis by the same experiment (dot-blot analysis), its expression there would also be expected to be below the sensitivity threshold of in situ hybridization. From the fact that antizyme 3 is expressed only in testis and then only in the spermatid phase, whereas antizymes 1 and 2 are only negligibly (if at all) expressed during spermatogenesis, it is concluded that antizyme 3 has evolved specifically to provide spatial and temporal regulation of ODC during spermatogenesis (Ivanov, 2000b).

The role of ODC and, by extension, polyamines in spermatogenesis is not clear. Several functions have been proposed. These functions include possible roles in DNA synthesis and packaging during meiosis or regulation of transcription in haploid spermatogenic cells. Whatever their roles in spermatogenesis, there is a good indication of what might be the physiological consequences of extending high levels of ODC expression past its normal stage in sperm development. The main phenotype of transgenic mice overproducing ODC is male infertility and an associated alteration in seminiferous epithelial morphology. In such animals there is a significant reduction of mature spermatozoa. In fact, this deleterious effect is observed in all postmeiotic cells. Perhaps, in these cells the normally occurring antizyme 3 is 'swamped out' by the extra ODC, which leads to excessive polyamine (putrescine) accumulation and premature cell death. If this hypothesis is correct, mammals (mice in particular) lacking this gene should be male infertile with a specific morphology of the seminiferous epithelium (i.e., normal Sertoli cells, spermatogonia, and spermatocytes, but reduced or absent spermatids and spermatozoa). If this phenotype is confirmed, the antizyme 3 gene will become a candidate for heritable forms of human male infertility with similar testicular morphology (certain types of Sertoli cell only syndrome). These findings are consistent with the conclusion that polyamines play an important and special role during late meiosis and early spermiogenesis (Ivanov, 2000b).


REFERENCES

Search PubMed for articles about Drosophila Ornithine decarboxylase antizyme

Almrud, J. J., et al. (2000). Crystal structure of human ornithine decarboxylase at 2.1 A resolution: structural insights to antizyme binding. J. Mol. Biol. 295(1): 7-16. 10623504

Chattopadhyay, M. K., Murakami, Y. and Matsufuji, S. (2001). Antizyme regulates the degradation of ornithine decarboxylase in fission yeast Schizosaccharomyces pombe. Study in the spe2 knockout strains. J. Biol. Chem. 276(24): 21235-41. 11283013

Chen, H., MacDonald, A. and Coffino, P. (2003). Structural elements of antizymes 1 and 2 are required for proteasomal degradation of ornithine decarboxylase. J. Biol. Chem. 277(48): 45957-61. 12359729

Coffino, P. (2001a). Regulation of cellular polyamines by antizyme. Nat. Rev. Mol. Cell Biol. 2(3): 188-94. 11265248

Coffino, P. (2001b). Antizyme, a mediator of ubiquitin-independent proteasomal degradation. Biochimie 83: 319-323. 11295492

Hoyt, M. A., Zhang, M. and Coffino, P. (2003). Ubiquitin-independent Mechanisms of Mouse Ornithine Decarboxylase Degradation Are Conserved between Mammalian and Fungal Cells. J. Biol. Chem. 278(14): 12135-43. 12562772

Gruendler, C., Lin, Y., Farley, J. and Wang, T. (2001). Proteasomal degradation of Smad1 induced by bone morphogenetic proteins. J. Biol. Chem. 276: 46533-46543. 11571290

Gupta. R., et al. (2001). Effect of spermidine on the in vivo degradation of ornithine decarboxylase in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. 98(19): 10620-3. 11535806

Ichiba, T., et al. (1994). Functional regions of ornithine decarboxylase antizyme. Biochem. Biophys. Res. Com. 200: 1721-1727. 8185631

Ivanov, I. P., Simin, K., Letsou, A., Atkins, J. F. and Gesteland, R. F. (1998). The Drosophila gene for antizyme requires ribosomal frameshifting for expression and contains an intronic gene for snRNP SmD3 on the opposite strand. Mol. Cell. Biol. 18: 1553-1561. 9488472

Ivanov, I. P., et al. (2000a). Conservation of polyamine regulation by translational frameshifting from yeast to mammals. EMBO J. 19(8): 1907-17. 10775274

Ivanov, I. P., Rohrwasser, A., Terreros, D., Gesteland, R. F. and Atkins, J. F. (2000b). Discovery of a spermatogenesis stage-specific ornithine decarboxylase antizyme: Antizyme 3. Proc. Natl. Acad. Sci. 97: 4808-4813. 10781085

Kania, A., et al. (1995). P-element mutations affecting embryonic peripheral nervous system development in Drosophila melanogaster. Genetics 139: 1663-1678. 7789767

Li, X., Stebbins, B., Hoffman, L., Pratt, G., Rechsteiner, M. and Coffino P. (1996). The N terminus of antizyme promotes degradation of heterologous proteins. J. Biol. Chem. 271(8): 4441-6. 8626796

Matsufuji, S., et al. (1995). Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell 80: 51-60. 7813017

Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K., Tamura, T., Tanaka, K., and Ichihara, A. (1992). Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature 360: 597-599. 1334232

Murakami, Y., Matsufuji, S., Hayashi, S. I., Tanahashi, N., and Tanaka, K. (1999). ATP-Dependent inactivation and sequestration of ornithine decarboxylase by the 26S proteasome are prerequisites for degradation. Mol. Cell. Biol. 19: 7216-7227. 10490656

Rom, E., and Kahana, C. (1994). Polyamines regulate the expression of ornithine decarboxylase antizyme in vitro by inducing ribosomal frame-shifting. Proc. Natl. Acad. Sci. 91: 3959-3963. 8171019

Salzberg, A., Golden, K., Bodmer, R. and Bellen, H. J. (1996). gutfeeling, a Drosophila gene encoding an Antizyme-like protein, is required for late differentiation of neurons and muscles. Genetics 144: 183-196

Satriano, J., et al. (1998). Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels. J. Biol. Chem. 273(25): 15313-6. 9624108

Schenkel, H., Hanke, S., De Lorenzo, C., Schmitt, R. and Mechler, B. M. (2002). P elements inserted in the vicinity of or within the Drosophila snRNP SmD3 gene nested in the first intron of the Ornithine Decarboxylase Antizyme gene affect only the expression of SmD3. Genetics 161: 763-772. PubMed ID: 12072471

Scorcioni, F., Corti, A., Davalli, P., Astancolle, S. and Bettuzzi, S. (2001). Manipulation of the expression of regulatory genes of polyamine metabolism results in specific alterations of the cell-cycle progression. Biochem. J. 354(Pt 1): 217-23. 11171097

Vied, C. and Horabin, J. I. (2001). The sex determination master switch, Sex lethal, responds to Hedgehog signaling in the Drosophila germline. Development 128: 2649-2660. 11526072

Vied, C., Halachmi, N., Salzberg, A. and Horabin, J. I. (2003). Antizyme is a target of Sex-lethal in the Drosophila germline and appears to act downstream of Hedgehog to regulate Sex-lethal and Cyclin B. Dev. Bio. 253: 214-229. 12645926

Zhang, M., Pickart, C. M. and Coffino, P. (2003). Determinants of proteasome recognition of ornithine decarboxylase, a ubiquitin-independent substrate. EMBO J. 22(7): 1488-96. 12660156

Zhu, C., Lang, D. W. and Coffino, P. (1999). Antizyme2 is a negative regulator of ornithine decarboxylase and polyamine transport. J. Biol. Chem. 274(37): 26425-30. 10473601


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