Depressive symptoms are associated with food insufficiency and nutritional deficiencies in poor community-dwelling elderly people.

UNLABELLED: Depression is associated with nutritional deterioration in older persons and is highly prevalent among people of low socioeconomic status (LSES). OBJECTIVES: To determine the prevalence of depressive symptoms and food insufficiency, and to examine the relationship between dietary intake, food insufficiency and depression, in LSES community dwelling elderly. DESIGN: Cross-sectional study. SETTING: Lod, a town in the central Israel. PARTICIPANTS: Community-dwelling welfare recipients aged 60 to 92. MEASUREMENTS: Depression was assessed by 15-item Geriatric Depression Scale (GDS-short version), using a score ≥ 10 as the cut off point for clinically important depressive symptoms. Dietary intake was evaluated using a 24-hour dietary recall. Food insufficiency was defined by participants reporting that they did not have enough food to eat " sometimes " or " often " . RESULTS: This study reports on 112 persons aged 60 years and above (27.1% men). The prevalence of depression in this population was 47%; 25% of the study sample was classified as " food insufficient " . Macronutrients intake was similar for depressed and non-depressed persons, except for polyunsaturated fats which was lower among the depressed group (7.9 ± 4.9 vs.11.0 ± 7.5 g/day in the non-depressed, p=0.03). Vitamins and minerals intake was lower than recommended for both groups; vitamin E intake was associated with depression. In regression models controlling for confounding variables, an increase of 1 mg in vitamin E intake and 1 gram in polyunsaturated fatty acids (PUFA) intake was associated with lower risk for depression (OR=0.73, p=0.008 and OR=0.86, p=0.007 respectively) Participants who reported food insufficiency were 10 times more likely to be depressed compared with those who reported sufficient food. CONCLUSIONS: Given the evaluated adverse association between depressive symptoms and food insufficiency, more efforts are needed to guarantee adequate food intake, particularly foods rich in vitamin E and PUFA, in poor elderly people. Further studies are needed to clarify the temporal relationship between the emotional and nutritional domains in this vulnerable population.

Ubiquitin dependent and independent protein degradation in the regulation of cellular polyamines.

Protein degradation mediated by the ubiquitin/proteasome system is the major route for the degradation of cellular proteins. In this pathway the ubiquitination of the target proteins is manifested via the concerted action of several enzymes. The ubiquinated proteins are then recognized and degraded by the 26S proteasome. There are few reports of proteins degraded by the 26S protesome without ubiquitination, with ornithine decarboxylase being the most notable representative of this group. Interestingly, while the degradation of ODC is independent of ubiquitination, the degradation of other enzymes of the polyamine biosynthesis pathway is ubiquitin dependent. The present review describes the degradation of enzymes and regulators of the polyamine biosynthesis pathway.

Antizyme inhibitor: a defective ornithine decarboxylase or a physiological regulator of polyamine biosynthesis and cellular proliferation.

ODC (ornithine decarboxylase) is a central regulator of cellular polyamine synthesis. ODC is a highly regulated enzyme stimulated by a variety of growth-promoting stimuli. ODC overexpression leads to cellular transformation. Cellular ODC levels are determined at transcriptional and translational levels and by regulation of its degradation. Here we review the mechanism of ODC degradation with particular emphasis on AzI (antizyme inhibitor), an ODC homologous protein that appears as a central regulator of ODC stability, cellular polyamine homoeostasis and cellular proliferation.

Overexpression of antizyme-inhibitor in NIH3T3 fibroblasts provides growth advantage through neutralization of antizyme functions.

Antizyme inhibitor (AzI) is a homolog of ornithine decarboxylase (ODC), a key enzyme of polyamine synthesis. Antizyme inhibitor retains no enzymatic activity, but exhibits high affinity to antizyme (Az), a negative regulator of polyamine homeostasis. As polyamines are involved in maintaining cellular proliferation, and since AzI may negate Az functions, we have investigated the role of AzI in regulating cell growth. We show here that overexpression of AzI in NIH3T3 cells increased growth rate, enabled growth in low serum, and permitted anchorage-independent growth in soft agar, while reduction of AzI levels by AzI siRNA reduced cellular proliferation. Moreover, AzI overproducing cells gave rise to tumors when injected into nude mice. AzI overexpression resulted in elevation of ODC activity and of polyamine uptake. These effects of AzI are a result of its ability to neutralize Az, as overexpression of an AzI mutant with reduced Az binding failed to alter cellular polyamine metabolism and growth properties. We also demonstrate upregulation of AzI in Ras transformed cells, suggesting its relevance to some naturally occurring transformations. Finally, increased uptake activity rendered AzI overproducing and Ras-transformed cells more sensitive to toxic polyamine analogs. Our results therefore imply that AzI has a central and meaningful role in modulation of polyamine homeostasis, and in regulating cellular proliferation and transformation properties.

Mechanism of polyamine tolerance in yeast: novel regulators and insights.

Polyamines are small charged molecules essential for various cellular functions, but at high levels they are cytotoxic. Two yeast kinases, SKY1 and PTK2, have been demonstrated to regulate polyamine tolerance. Here we report the identification and characterization of additional genes involved in regulating polyamine tolerance: YGL007W, FES1 and AGP2. Deletion of YGL007W, an open reading frame located within the promoter of the membrane proton pump PMA1, decreased Pma1p expression. Deletion of FES1 or AGP2 resulted in reduced polyamine uptake. While high-affinity spermine uptake was practically absent in agp2Delta cells, fes1Delta cells displayed only reduced affinity towards spermine. Despite the reduced uptake, the resistant strains accumulated significant levels of polyamines and displayed increased ornithine decarboxylase activity, suggesting reduced polyamine sensing. Interestingly, fes1Delta cells were highly sensitive to salt ions, suggesting different underlying mechanisms. These results indicate that mechanisms leading to polyamine tolerance are complex, and involve components other than uptake.

Mechanisms of ubiquitin-mediated, limited processing of the NF-kappaB1 precursor protein p105.

In most cases, target proteins of the ubiquitin system are completely degraded. In several exceptions, such as the first step in the activation of the transcriptional regulator NF-kappaB, the substrate, the precursor protein p105, is processed in a limited manner to yield the active subunit p50. p50 is derived from the N-terminal domain of p105, whereas the C-terminal domain is degraded. The mechanisms involved in this unique process have remained elusive. We have shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is one important processing signal and that it interferes with processing of the ubiquitinated precursor by the 26S proteasome. Also, amino acid residues 441-454 are important for processing under non-stimulated conditions. Lys 441 and 442 serve as ubiquitination targets, whereas residues 446-454 may serve as a ligase recognition motif. Following IkappaB kinase (IKK)-mediated phosphorylation, the C-terminal domain of p105, residues 918-934, recruits the SCF(beta-TrCP) ubiquitin ligase, and ubiquitination by this complex leads to accelerated processing. The two sites appear to be recognized under different physiological conditions by two different ligases, targeting two distinct recognition motifs. We have shown that ubiquitin conjugation and processing of a series of precursors of p105 that lack the C-terminal IKK phosphorylation/TrCP binding domain, is progressively inhibited with increasing number of ankyrin repeats. Inhibition is due to docking of active NF-kappaB subunits to the ankyrin repeat domain in the C-terminal half of p105 (IkappaBgamma). Inhibition is alleviated by phosphorylation of the C-terminal domain that leads to ubiquitin-mediated degradation of the ankyrin repeat domain and release of the anchored subunits. We propose a model that may explain the requirement for two sites: a) a basal site that may be involved in co-translational processing prior to the synthesis of the ankyrin repeat domain; and b) a signal-induced site that is involved in processing/degradation of the complete molecule following cell activation, with rapid release of stored, transcriptionally active subunits.

Screening for modulators of spermine tolerance identifies Sky1, the SR protein kinase of Saccharomyces cerevisiae, as a regulator of polyamine transport and ion homeostasis.

Although most cells are capable of transporting polyamines, the mechanism that regulates polyamine transport in eukaryotes is still largely unknown. Using a genetic screen for clones capable of restoring spermine sensitivity to spermine-tolerant mutants of Saccharomyces cerevisiae, we have demonstrated that Sky1p, a recently identified SR protein kinase, is a key regulator of polyamine transport. Yeast cells deleted for SKY1 developed tolerance to toxic levels of spermine, while overexpression of Sky1p in wild-type cells increased their sensitivity to spermine. Expression of the wild-type Sky1p but not of a catalytically inactive mutant restored sensitivity to spermine. SKY1 disruption results in dramatically reduced uptake of spermine, spermidine, and putrescine. In addition to spermine tolerance, sky1Delta cells exhibit increased tolerance to lithium and sodium ions but somewhat increased sensitivity to osmotic shock. The observed halotolerance suggests potential regulatory interaction between the transport of polyamines and inorganic ions, as suggested in the case of the Ptk2p, a recently described regulator of polyamine transport. We demonstrate that these two kinases act in two different signaling pathways. While deletion or overexpression of SKY1 did not significantly affect Pma1p activity, the ability of overexpressed Sky1p, Ptk1p, and Ptk2p to increase sensitivity to LiCl depends on the integrity of PPZ1 but not of ENA1.

Poliovirus 2A protease induces apoptotic cell death.

A cell line was generated that expresses the poliovirus 2A protease in an inducible manner. Tightly controlled expression was achieved by utilizing the muristerone A-regulated expression system. Upon induction, cleavage of the eukaryotic translation initiation factor 4GI (eIF4GI) and eIF4GII is observed, with the latter being cleaved in a somewhat slower kinetics. eIF4G cleavage was accompanied by a severe inhibition of protein synthesis activity. Upon induction of the poliovirus 2A protease, the cells displayed fragmented nuclei, chromatin condensation, oligonucleosome-size DNA ladder, and positive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) staining; hence, their death can be characterized as apoptosis. These results indicate that the expression of the 2A protease in mammalian cells is sufficient to induce apoptosis. We suggest that the poliovirus 2A protease induces apoptosis either by arresting cap-dependent translation of some cellular mRNAs that encode proteins required for cell viability, by preferential cap-independent translation of cellular mRNAs encoding apoptosis inducing proteins, or by cleaving other, yet unidentified cellular target proteins.

Structural motifs involved in ubiquitin-mediated processing of the NF-kappaB precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain.

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-kappaB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.

Insulin induces transcription of target genes through the hypoxia-inducible factor HIF-1alpha/ARNT.

Hypoxic stress induces the expression of genes associated with increased energy flux, including the glucose transporters Glut1 and Glut3, several glycolytic enzymes, nitric oxide synthase, tyrosine hydroxylase, erythropoietin and vascular endothelial growth factor (VEGF). Induction of these genes is mediated by a common basic helix-loop-helix-PAS transcription complex, the hypoxia-inducible factor-1alpha (HIF-1alpha)/aryl hydrocarbon nuclear translocator (ARNT). Insulin also induces some of these genes; however, the underlying mechanism is unestablished. We report here that insulin shares with hypoxia the ability to induce the HIF-1alpha/ARNT transcription complex in various cell types. This induction was demonstrated by electrophoretic mobility shift of the hypoxia response element (HRE), and abolished by specific antisera to HIF-1alpha and ARNT, and by transcription activation of HRE reporter vectors. Furthermore, basal and insulin-induced expression of Glut1, Glut3, aldolase A, phosphoglycerate kinase and VEGF was reduced in cells having a defective ARNT. Similarly, the insulin-induced activation of HRE reporter vectors and VEGF was impaired in these cells and was rescued by re-introduction of ARNT. Finally, insulin-like growth factor-I (IGF-I) also induced the HIF-1alpha/ARNT transcription complex. These observations establish a novel signal transduction pathway of insulin and IGF-I and broaden considerably the scope of activity of HIF-1alpha/ARNT.

Basal and human papillomavirus E6 oncoprotein-induced degradation of Myc proteins by the ubiquitin pathway.

We have previously shown that the degradation of c-myc and N-myc in vitro is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins in vivo and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins in cells is inhibited by lactacystin and MG132, two inhibitors of the 20S proteasome. Inhibition is accompanied by accumulation of myc-ubiquitin conjugates. Dissection of the ancillary proteins involved revealed that the high-risk human papillomavirus oncoprotein E6-16 stimulates conjugation and subsequent degradation of the myc proteins in vitro. Expression of E6-16 in cells results in significant shortening of the t1/2 of the myc proteins with subsequent decrease in their cellular level. Analysis of the conjugating enzymes revealed that under basal conditions the proteins can be conjugated by two pairs of E2s and E3s-E2-14 kDa and E3alpha involved in the "N-end rule" pathway, and E2-F1 (UbcH7) and E3-Fos involved also in conjugation of c-Fos. In the presence of E6-16, a third pair, E2-F1 and E6-AP mediate conjugation of myc by means of a mechanism that appears to be similar to that involved in the targeting of p53, formation of a myc. E6.E6-AP targeting complex. It is possible that in certain cells E6-mediated targeting of myc prevents myc-induced apoptosis and thus ensures maintenance of viral infection.

c-Myc and Max transregulate the mouse ornithine decarboxylase promoter through interaction with two downstream CACGTG motifs.

Ornithine decarboxylase (ODC), the first enzyme in the biosynthesis of polyamines, is essential for the process of cellular proliferation. ODC is a typical delayed early gene, as its mitogenic activation requires ongoing protein synthesis in the stimulated cells. This study provides evidence that the immediate early c-Myc protein is a potential transactivator of the ODC gene. We demonstrate that overexpression of c-Myc results in efficient activation of the ODC promoter, whereas overexpression of Max exerts a repressive effect. Both effects depend on the presence of two evolutionary conserved CACGTG motifs found in the first intron of the ODC gene. Transactivation of the ODC promoter also requires the dimerization of c-Myc with Max. Interestingly, over-expression of USF, a member of the same family of proteins which efficiently binds these two CACGTG motifs, fails to transregulate the ODC promoter. Our data suggest that c-Myc and Max are potential transcriptional regulators of the ODC promoter.

Exposure to ornithine results in excessive accumulation of putrescine and apoptotic cell death in ornithine decarboxylase overproducing mouse myeloma cells.

Ornithine decarboxylase (ODC) is the first key enzyme in the biosynthesis of polyamines, aliphatic polycations that are indispensable for the process of mammalian cell proliferation. The mouse myeloma cell line, 653-1, massively overproduces ODC due to the amplification of an active ODC gene. The addition of ornithine to the growth medium of 653-1 cells results in a massive increase in the intracellular concentration of putrescine, followed by rapid cell death. Ornithine-treated 653-1 cells display fragmented nuclei, chromatin condensation, and an oligonucleosome-sized DNA "ladder"; consequently, their death can be described as apoptosis. Accumulation of putrescine in 653-1 cells is accompanied by a rapid decrease of protein synthesis activity, suggesting that protein synthesis inhibition may be the cause for the apoptotic death of 653-1 cells. However, since the apoptotic death provoked by exposure of 653-1 cells to ornithine reached a maximal level earlier than that caused by cycloheximide, we conclude that protein synthesis inhibition is unlikely to be the direct cause of the observed apoptotic cell death.

Degradation of ornithine decarboxylase by the mammalian and yeast 26S proteasome complexes requires all the components of the protease.

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is an extremely short-lived protein. This attribute is important for the regulation of the activity of the enzyme and implies that the mechanisms involved in its degradation play an important role in the control of the cellular processes in which the enzyme is involved. Recently, it has been shown that ODC is degraded by the 26S proteasome complex in a process that requires antizyme, but not ubiquitin. With one reported exception, ODC, the 26S complex recognizes and degrades specifically ubiquitinated proteins. Their unconjugated counterparts are not targeted. The 26S complex is composed of a core catalytic unit, the 20S proteasome complex, and two additional, and apparently distinct, subcomplexes. The two additional subcomplexes are regulatory subunits that are required in order to confer specificity and control. In this study, we demonstrate that, like the degradation of ubiquitin-conjugated proteins, ubiquitin-independent degradation of ODC also requires prior assembly of the mammalian 26S proteasome from all the three subunits, the 20S proteasome and the two subcomplexes. The combination of any two subunits does not support generation of a proteolytically active complex. This is also true for the yeast 26S complex. Like the mammalian 20S proteasome, the yeast 20S complex can cleave short peptides in an ATP-independent mode, but cannot degrade ODC or ubiquitin-conjugated proteins. These proteins are degraded only following addition of the regulatory subunits and generation of the high-molecular-mass 26S complex. In a distinct, but related, set of experiments, we demonstrate that the degradation of ODC by the assembled 26S proteasome in vitro reproduces faithfully proteolysis of the enzyme in the intact cell. Namely, (a) a C-terminal-deleted mouse ODC and trypanosome ODC are stable both in vitro and in vivo, and (b) like proteolysis in the intact cell, degradation in the reconstituted cell-free system is also dependent upon the addition of antizyme.

The 26S proteasome degrades mouse and yeast ornithine decarboxylase in yeast cells.

Eukaryotic cells possess two high-molecular-mass proteases, the 700 kDa, 20S proteasome, as well as the even larger 1,400 kDa, 26S proteasome. It has been demonstrated that ornithine decarboxylase is degraded, in vitro, by the 26S proteasome that contains the 20S protease as its catalytic core, but not by the free 20S proteasome. Recently, by demonstrating severe inhibition of mouse and yeast ODC degradation in a mutant yeast cell line, defective in the chymotripsin-like activity of the yeast 20S proteasome, we implicated the 20S proteasome in the degradation of ODC, in vivo, in yeast cells. Here we show that the degradation of ODC is also severely inhibited in the mutant yeast cell lines, cim3-1 and cim5-1, containing a specific lesion in subunits that are unique to the yeast 26S proteasome. We therefore, conclude, that as illustrated in vitro, also in intact cells, it is the 26S proteasome, not the free 20S proteasome, that degrades ODC. We also demonstrate, that while deficiency in the proteasome chymotrypsine-like activity (in the yeast pre1-1 mutant) inhibits the degradation of both yeast and mouse ODCs, deficiency in the peptidyl-glutamyl-peptide-hydrolyzing (PGPH) activity inhibits only yeast ODC degradation. Similarly, we have noted that whereas the putative ATPase activity of both the CIM3 and CIM5 subunits is essential for the degradation of mouse ODC, only that of the CIM3 subunit is required for the degradation of yeast ODC. These results suggest differential utilization of individual proteasomal subunits in the recognition and degradation of individual short-lived proteins.

A unified pathway for the degradation of ornithine decarboxylase in reticulocyte lysate requires interaction with the polyamine-induced protein, ornithine decarboxylase antizyme.

Recent studies have provided convincing evidence to add to a number of earlier observations suggesting that the rapid intracellular degradation of mammalian ornithine decarboxylase (ODC) is further accelerated by the action of ornithine decarboxylase antizyme (ODC-Az), a polyamine-induced protein. However, the mechanism whereby ODC-Az exerts its effect in this proteolytic process is mostly unknown. Here, by using reticulocyte-lysate-based synthesis and degradation systems, we demonstrate that interaction of ODC-Az with ODC results in two related outcomes: (a) ODC is inactivated as a result of its monomerization, and (b) ODC degradation is dramatically accelerated. While ODC inactivation requires the integrity of the ODC-Az binding site of ODC and the ODC binding site of ODC-Az, acceleration in ODC degradation also requires the previously characterized carboxyl-terminal destabilizing segment of ODC and a specific segment of ODC-Az that may be functionally distinct from that required for ODC binding. Interestingly, an active ODC variant with a mutant ODC-Az binding site is stable under basal degradation conditions. This, together with the ability of anti-(ODC-Az) antibody to specifically inhibit the basal degradation of ODC in the lysate, suggests that ODC-Az is an essential general mediator of ODC degradation. Based on these observations, we propose a model for the degradation of ODC which always require interaction with antizyme.

Protein synthesis elongation factor EF-1 alpha is essential for ubiquitin-dependent degradation of certain N alpha-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu.

Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin pathway involves diverse recognition signals and distinct enzymatic factors. A few proteins are recognized via their N-terminal amino acid residue and conjugated by a ubiquitin-protein ligase that recognizes this residue. Most substrates, including the N alpha-acetylated proteins that constitute the vast majority of cellular proteins, are targeted by different signals and are recognized by yet unknown ligases. We have previously shown that degradation of N-terminally blocked proteins requires a specific factor, designated FH, and that the factor acts along with the 26S protease complex to degrade ubiquitin-conjugated proteins. Here, we demonstrate that FH is the protein synthesis elongation factor EF-1 alpha. (a) Partial sequence analysis reveals 100% identity to EF-1 alpha. (b) Like EF-1 alpha, FH binds to immobilized GTP (or GDP) and can be purified in one step using the corresponding nucleotide for elution. (c) Guanine nucleotides that bind to EF-1 alpha protect the ubiquitin system-related activity of FH from heat inactivation, and nucleotides that do not bind do not exert this effect. (d) EF-Tu, the homologous bacterial elongation factor, can substitute for FH/EF-1 alpha in the proteolytic system. This last finding is of particular interest since the ubiquitin system has not been identified in prokaryotes. The activities of both EF-1 alpha and EF-Tu are strongly and specifically inhibited by ubiquitin-aldehyde, a specific inhibitor of ubiquitin isopeptidases. It appears, therefore, that EF-1 alpha may be involved in releasing ubiquitin from multiubiquitin chains, thus rendering the conjugates susceptible to the action of the 26S protease complex.

Polyamines regulate the expression of ornithine decarboxylase antizyme in vitro by inducing ribosomal frame-shifting.

We provide here an example of a mammalian cellular gene expressed by frame-shifting. 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. By translating, in vitro in reticulocyte lysate, antizyme mRNA with a full coding capacity and various mutants derived from it, we demonstrate that antizyme expression requires that ribosomes shift from the first open reading frame (termed ORF0) to a second +1 open reading frame (ORF1). Our 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, we have shown that an 87-nt segment surrounding stop-1 enhances frame-shifting efficiency, whereas the 6 nt located just upstream to stop-1 are absolutely essential for this process. Because this segment does not contain sequences that were previously characterized as shifty segments, our results suggest that another mechanism of frame-shifting is involved in mediating antizyme expression.

The 20S proteasome mediates the degradation of mouse and yeast ornithine decarboxylase in yeast cells.

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is one of the most rapidly degraded proteins in mammalian cells. Recently it has been demonstrated that mammalian ODC is degraded in vitro by the 26S protease that contains the 20S proteasome as its catalytic core, in a reaction that does not require ubiquitin. Here, we show that yeast and mouse ODC are both rapidly degraded in yeast cells and that their degradation severely inhibited in a mutant yeast cell line defective in the chymotryptic activity of proteinase yscE, the yeast 20S proteasome. These results provide compelling genetic support to previous biochemical studies suggesting the involvement of the 20S proteasome in the degradation of ornithine decarboxylase.

Gly387 of murine ornithine decarboxylase is essential for the formation of stable homodimers.

In its active form mammalian ornithine decarboxylase (ODC) is a homodimer composed of two 53-kDa subunits while the monomer retains no enzymic activity. In the present study we demonstrate that Gly387 of mouse ODC plays an important role in enabling dimer formation. Gly387 of mouse ODC, an evolutionary conserved residue, was converted to all possible 19 amino acids using site-directed mutagenesis. With the exception of alanine, all other substitutions of Gly387 completely abolished enzymic activity. Cross-linking analysis and fractionation through a Superose-12 sizing column have demonstrated that mutant subunits are detected only in their monomeric form. These results strongly suggest that the primary lesion of substitution at position 387 of mouse ODC is the inability of mutant subunits to associate with each other to form the active homodimers. In agreement with this conclusion, G387A, the only mutant that retained partial activity, displayed reduced dimerization. The degradation rate of ODC mutants in which Gly387 was substituted by aspartic acid or alanine was enhanced compared to the wild-type enzyme, suggesting that monomers may be more susceptible to degradation.