In vitro study of proteolytic degradation of rat histidine decarboxylase.

Mammalian ornithine decarboxylase (ODC) is a very unstable protein which is degraded in an ATP-dependent manner by proteasome 26S, after making contact with the regulatory protein antizyme. PEST regions are sequences described as signals for protein degradation. The C-terminal PEST region of mammalian ODC is essential for its degradation by proteasome 26S. Mammalian histidine decarboxylase (HDC) is also a short-lived protein. The full primary sequence of mammalian HDC contains PEST-regions at both the N- and C-termini. Rat ODC and different truncated and full versions of rat HDC were expressed in vitro. In vitro degradation of rat ODC and rat 1-512 HDC were compared. Like ODC, rat 1-512 HDC is degraded mainly by an ATP-dependent mechanism. However, antizyme has no effect on the degradation of 1-512 HDC. The use of the inhibitors MG-132 and lactacystine significantly inhibited the degradation of 1-512 HDC, suggesting that a ubiquitin-dependent, proteasome 26S proteolytic pathway is involved. Results obtained with the different modifications of rat HDC containing all three PEST regions (full version, 1-656 HDC), only the N-terminal PEST region (1-512 HDC), or no PEST region (69-512 HDC), indicate that the N-terminal (1-69) fragment, but not the C-terminal fragment, determines that the HDC protein is a proteasome substrate in vitro.     

Solution structure of a conserved domain of antizyme: a protein regulator of polyamines

Antizyme and its isoforms are members of an unusual yet broadly conserved family of proteins, with roles in regulating polyamine levels within cells. Antizyme has the ability to bind and inhibit the enzyme ornithine decarboxylase (ODC), targeting it for degradation at the proteasome; antizyme is also known to affect the transport of polyamines and interact with the antizyme inhibitor protein (AZI), as well as the cell-cycle protein cyclin D1. In the present work, NMR methods were used to determine the solution structure of a stable, folded domain of mammalian antizyme isoform-1 (AZ-1), consisting of amino acid residues 87-227. The protein was found to contain eight beta strands and two alpha helices, with the strands forming a mixed parallel and antiparallel beta sheet. At the level of primary sequence, antizyme is not similar to any protein of known structure, and results show that antizyme exhibits a novel arrangement of its strands and helices. Interestingly, however, the fold of antizyme is similar to that found in a family of acetyl transferases, as well as translation initiation factor IF3, despite a lack of functional relatedness between these proteins. Structural results, combined with amino acid sequence comparisons, were used to identify conserved features among the various homologues of antizyme and their isoforms. Conserved surface residues, including a cluster of acidic amino acids, were found to be located on a single face of antizyme, suggesting this surface is a possible site of interaction with target proteins such as ODC. This structural model provides an essential framework for an improved future understanding of how the different parts of antizyme play their roles in polyamine regulation.     

Degrons of yeast and mammalian ornithine decarboxylase enzymes make potent combination for regulated targeted protein degradation

Elevation of polyamine levels in eukaryotes leads to rapid degradation of ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis pathway. ODC in yeast (yODC) has two domains, the Nα/β domain consisting of α/β barrel domain (α/β) preceded by an overhang of 50 residues at its N-terminus (N50) and β sheet domain at its C-terminus. Two degradation determinant signals or degrons in yODC sequence, namely the N50 and the antizyme-binding element (AzBE) housed in the α/β domain, are responsible for its degradation by proteasomes. Antizyme (Az) induced under polyamine excess binds to AzBE and delivers ODC to proteasome, while the N50 threads the protein into proteasome. It was previously reported by us that the peptide Nα/β of yODC acts as an independent transplantable degron, whose action can be modulated with the help of antizyme by varying polyamine levels. Mammalian ODC (mODC), in spite of its 40% sequence homology with yODC, is devoid of N50 of yODC and instead sports a C-terminal tail of 37 residues (CmODC). CmODC acts as an independent transplantable degron with no equivalent in yODC. The present study investigates the merits of employing the two degrons Nα/β and CmODC together for targeted protein degradation by expressing them in a chimeric fusion with green fluorescent protein (GFP). Our results establish that under the regulation of antizyme, the signals Nα/β and CmODC acting together enhance degradation better than either degron in isolation. The combination of Nα/β and CmODC can be employed to study the function of novel proteins through their rapid removal.

     

Histidine decarboxylase gene expression in rat fundus is regulated by gastrin

The conversion of histidine to histamine by histidine decarboxylase (HDC) is of central importance in the control of vertebrate acid secretion. We have used PCR-generated probes to study the regulation of HDC gene expression in rat fundic mucosa. When circulating gastrin levels were lowered by fasting or elevated by treatment with omeprazole, there were parallel changes in HDC mRNA abundance. However, when animals with elevated gastrin levels were concurrently treated with the gastrin/CCK-B receptor antagonist PD 134308, HDC mRNA levels were not increased. These data are consistent with the hypothesis that HDC gene expression is regulated by gastrin, over the physiological range of circulating hormone concentrations.     

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.     

PACAP and gastrin regulate the histidine decarboxylase promoter via distinct mechanisms

The enterochromaffin-like (ECL) cell controls gastric acid secretion via histamine, generated by l-histidine decarboxylase (HDC). HDC expression is regulated by gastrin. However, gastrin is not alone in controlling ECL cell function. For example, the neural peptide pituitary adenylate cyclase-activating polypeptide (PACAP) also increases ECL cell proliferation. To investigate a potential role of PACAP in regulating HDC expression, we generated a series of HDC promoter-luciferase reporter constructs and transiently transfected them into PC12 cells (stably expressing the gastrin-CCK-2 receptor). We found that PACAP regulates HDC promoter activity. This is temporally biphasic, involving both adenyl cyclase and phospholipase C-dependent pathways. Deletional analysis, block mutation, and EMSA demonstrated a PACAP-response element at -177 to -170, wholly necessary for the effects of PACAP and discrete from known gastrin-responsive elements. Discrete neural and endocrine pathways regulate ECL cells through different patterns of postreceptor signaling and promoter activation, which may be appropriate to their functions in vivo.     

抗酶的研究进展

抗酶（antizyme）是当细胞内多胺水平升高时刺激机体合成的一种小分子量调节蛋白,能特异性地与鸟氨酸脱羧酶（omithine decarboxylase,ODC）结合,经泛素非依赖途径被26S蛋白酶体降解,从而使多胺合成减少;抗酶还可以调节多胺转运,以稳定细胞内多胺水平。近年来随着生物技术的不断发展,对抗酶的认识也逐步深入,本文综述了抗酶家族、合成、作用及定位等方面的研究进展。     

The antizyme family: polyamines and beyond

The family of antizymes functions as regulators of polyamine homeostasis. They are a class of small, inhibitory proteins, whose expression is regulated by a unique ribosomal frameshift mechanism. They have been shown to inhibit cell proliferation and possess anti-tumor activity. Antizymes bind ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. They inhibit its enzymatic activity and promote the ubiquitin-independent degradation of ODC by the 26S proteasome. In addition, they also negatively regulate polyamine transport. Antizyme-mediated, ubiquitin-independent degradation of ODC is conserved from yeast to man. But recent data suggest that this degradation pathway might not be restricted to ODC alone and could involve newly discovered antizyme binding partners. Interestingly, antizyme proteins have been strictly preserved over a vast evolutionary timeframe. Antizymes consequently represent an important class of proteins that regulate cell growth and metabolism by a diverse set of mechanisms that include protein degradation, inhibition of enzyme activity, small molecule transport and other, potentially not yet discovered properties.     

Antizyme targets cyclin D1 for degradation. A novel mechanism for cell growth repression

Overproduction of the ornithine decarboxylase (ODC) regulatory protein ODC-antizyme has been shown to correlate with cell growth inhibition in a variety of different cell types. Although the exact mechanism of this growth inhibition is not known, it has been attributed to the effect of antizyme on polyamine metabolism. Antizyme binds directly to ODC, targeting ODC for ubiquitin-independent degradation by the 26 S proteasome. We now show that antizyme induction also leads to degradation of the cell cycle regulatory protein cyclin D1. We demonstrate that antizyme is capable of specific, noncovalent association with cyclin D1 and that this interaction accelerates cyclin D1 degradation in vitro in the presence of only antizyme, cyclin D1, purified 26 S proteasomes, and ATP. In vivo, antizyme up-regulation induced either by the polyamine spermine or by antizyme overexpression causes reduction of intracellular cyclin D1 levels. The antizyme-mediated pathway for cyclin D1 degradation is independent of the previously characterized phosphorylation- and ubiquitination-dependent pathway, because antizyme up-regulation induces the degradation of a cyclin D1 mutant (T286A) that abrogates its ubiquitination. We propose that antizyme-mediated degradation of cyclin D1 by the proteasome may provide an explanation for the repression of cell growth following antizyme up-regulation.     

Evidence of a role for antizyme and antizyme inhibitor as regulators of human cancer

Antizyme and its endogenous antizyme inhibitor have recently emerged as prominent regulators of cell growth, transformation, centrosome duplication, and tumorigenesis. Antizyme was originally isolated as a negative modulator of the enzyme ornithine decarboxylase (ODC), an essential component of the polyamine biosynthetic pathway. Antizyme binds ODC and facilitates proteasomal ODC degradation. Antizyme also facilitates degradation of a set of cell cycle regulatory proteins, including cyclin D1, Smad1, and Aurora A kinase, as well as Mps1, a protein that regulates centrosome duplication. Antizyme has been reported to function as a tumor suppressor and to negatively regulate tumor cell proliferation and transformation. Antizyme inhibitor binds to antizyme and suppresses its known functions, leading to increased polyamine synthesis, increased cell proliferation, and increased transformation and tumorigenesis. Gene array studies show antizyme inhibitor to be amplified in cancers of the ovary, breast, and prostate. In this review, we summarize the current literature on the role of antizyme and antizyme inhibitor in cancer, discuss how the ratio of antizyme to antizyme inhibitor can influence tumor growth, and suggest strategies to target this axis for tumor prevention and treatment.     

PEST sequences mediate heat shock factor 2 turnover by interacting with the Cul3 subunit of the Cul3-RING ubiquitin ligase

Cullin-RING ubiquitin ligases promote the polyubiquitination and degradation of many important cellular proteins, which previous studies indicated can be targeted for degradation via interaction with BTB domain-containing subunits of this E3 ligase complex. PEST domains are known to promote the degradation of proteins that contain them. However, the molecular mechanism by which PEST sequences promote degradation of these proteins is not understood. Here we show that the PEST sequences of a short-lived protein called HSF2 interact with Cullin3, a subunit of a Cullin-RING E3 ubiquitin ligase, and that this interaction mediates the Cul3-dependent ubiquitination and degradation of HSF2. These results indicate how, at the molecular level, PEST sequences can promote the proteolysis of proteins that contain them. They also expand understanding of the mechanisms by which substrates can be recruited to Cullin-RING E3 ubiquitin ligases to include interactions between PEST sequences and Cul3.     

Transferable domain in the G(1) cyclin Cln2 sufficient to switch degradation of Sic1 from the E3 ubiquitin ligase SCF(Cdc4) to SCF(Grr1)

Degradation of Saccharomyces cerevisiae G(1) cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 (SCF(Grr1)). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway.     

The mechanism of histamine secretion from gastric enterochromaffin-like cells

Enterochromaffin-like (ECL) cells play a pivotal role in theperipheral regulation of gastric acid secretion as they respond to thefunctionally important gastrointestinal hormones gastrin andsomatostatin and neural mediators such as pituitary adenylate cyclase-activating peptide and galanin. Gastrin is the keystimulus of histamine release from ECL cells in vivo and in vitro.Voltage-gated K⁺ andCa²⁺ channels have been detectedon isolated ECL cells. Exocytosis of histamine following gastrinstimulation and Ca²⁺ entry acrossthe plasma membrane is catalyzed by synaptobrevin andsynaptosomal-associated protein of 25 kDa, both characterized as asoluble N-ethylmaleimide-sensitivefactor attachment protein receptor protein. Histamine release occursfrom different cellular pools: preexisting vacuolar histamineimmediately released by Ca²⁺ entryor newly synthesized histamine following induction of histidine decarboxylase (HDC) by gastrin stimulation. Histamine is synthesized bycytoplasmic HDC and accumulated in secretory vesicles byproton-histamine countertransport via the vesicular monoaminetransporter subtype 2 (VMAT-2). The promoter region of HDC containsCa²⁺-, cAMP-, and protein kinaseC-responsive elements. The gene promoter for VMAT-2, however, lacksTATA boxes but contains regulatory elements for the hormones glucagonand somatostatin. Histamine secretion from ECL cells is thereby under acomplex regulation of hormonal signals and can be targeted at severalsteps during the process of exocytosis.

     

Ornithine decarboxylase antizyme Oaz3 modulates protein phosphatase activity

Ornithine decarboxylase antizyme 3 (Oaz3) is expressed in spermatids, makes up the antizyme family of Oaz genes with Oaz1 and Oaz2, and was proposed to encode a 22 kDa antizyme protein involved in polyamine regulation similar to the 22 kDa OAZ1 and OAZ2 proteins. Here we demonstrate however that the major product encoded by Oaz3 is a 12 kDa protein, p12, which lacks the antizyme domain that interacts with ornithine decarboxylase. We show that p12 does not affect ornithine decarboxylase levels, providing an explanation for the surprising observation made in Oaz3 knock-out male mice, which do not display altered testis polyamine metabolism. This suggested a novel activity for Oaz3 p12. Using immuno-electron microscopy we localized p12 to two structures in the mammalian sperm tail, viz. the outer dense fibers and fibrous sheath, as well as to the connecting piece linking head and tail. We identified myosin phosphatase targeting subunit 3 (MYPT3), a regulator of protein phosphatase PP1β, as a major p12-interacting protein, and show that MYPT3 is present in sperm tails and that its ankyrin repeat binds p12. We show that MYPT3 can also bind protein phosphatase PP1γ2, the only protein phosphatase present in sperm tails, and that p12- MYPT3 interaction modulates the activity of both PP1β and PP1γ2. This is, to our knowledge, the first demonstration of a novel activity for an Oaz-encoded protein.     

Involvement of antizyme in stabilization of ornithine decarboxylase caused by inhibitors of polyamine synthesis

Contrary to previous findings, ornithine decarboxylase (ODC) was stabilized by treatment of cells with DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ODC. Both this inhibitor and cyclohexylamine, a spermidine synthase inhibitor known to stabilize ODC, caused decreases in the antizyme/ODC ratio by increasing ODC content and conversely decreasing antizyme content. The relationship between cellular polyamine levels and antizyme content indicated that spermidine is the most important polyamine for antizyme induction. These results suggest that antizyme is involved in the mechanism underlying the stabilization of ODC by inhibitors of polyamine synthesis and support the hypothesis that cellular polyamines regulate ODC degradation via antizyme.     

Antizyme affects cell proliferation and viability solely through regulating cellular polyamines

Antizymes are key regulators of cellular polyamine metabolism that negatively regulate cell proliferation and are therefore regarded as tumor suppressors. Although the regulation of antizyme (Az) synthesis by polyamines and the ability of Az to regulate cellular polyamine levels suggest the centrality of polyamine metabolism to its antiproliferative function, recent studies have suggested that antizymes might also regulate cell proliferation by targeting to degradation proteins that do not belong to the cellular polyamine metabolic pathway. Using a co-degradation assay, we show here that, although they efficiently stimulated the degradation of ornithine decarboxylase (ODC), Az1 and Az2 did not affect or had a negligible effect on the degradation of cyclin D1, Aurora-A, and a p73 variant lacking the N-terminal transactivation domain whose degradation was reported recently to be stimulated by Az1. Furthermore, we demonstrate that, although Az1 and Az2 could not be constitutively expressed in transfected cells, they could be stably expressed in cells that express trypanosome ODC, a form of ODC that does not bind Az and therefore maintains a constant level of cellular polyamines. Taken together, our results clearly demonstrate that Az1 and Az2 affect cell proliferation and viability solely by modulating cellular polyamine metabolism.     

A novel route for F-box protein-mediated ubiquitination links CHIP to glycoprotein quality control

In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif.