Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:4.1.1.17 (ornithine decarboxylase)
6,351 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The half-life of the green fluorescent protein (GFP) was determined biochemically in cultured mouse LA-9 cells. The wild-type protein was found to be stable with a half-life of approximately 26 h, but could be destabilized by the addition of putative proteolytic signal sequences derived from proteins with shorter half-lives. A C-terminal fusion of a PEST sequence from the mouse ornithine decarboxylase gene reduced the half-life to 9.8 h, resulting in a GFP variant suitable for the study of dynamic cellular processes. In an N-terminal fusion containing the mouse cyclin B1 destruction box, it was reduced to 5.8 h, with most degradation taking place at metaphase. The combination of both sequences produced a similar GFP half-life of 5.5 h. Thus, the stability of this marker protein can be controlled in predetermined ways by addition of the appropriate proteolytic signals.
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PMID:Attenuation of green fluorescent protein half-life in mammalian cells. 1061 96

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.
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PMID:In vitro study of proteolytic degradation of rat histidine decarboxylase. 1069 92

The ornithine decarboxylase (ODC) gene of the human respiratory fungal pathogen, Coccidioides immitis (Ci) was cloned, sequenced, chromosome-mapped, and expressed in Escherichia coli (Ec). The genomic, cDNA and translated sequences are presented. Transformation of an ODC null mutant strain of Ec (EWH 319) with the Ci ODC gene was conducted to confirm function of the protein encoded by the fungal gene. Activity of the enzyme by the bacterial transformant was inhibited by 1, 4-diamino-2-butanone (DAB), a known inhibitor of eukaryotic ODC. Temporal expression of the Ci ODC gene during the parasitic cell cycle is constitutive, based on results of RT PCR. However, results of enzyme activity assays of cell homogenates obtained at different stages of parasitic cell development in vitro showed that the functional protein is present only during periods of isotropic growth and segmentation, and these morphogenetic events can be arrested by the addition of DAB. The observed absence of a difference in steady-state mRNA transcript amounts, and the developmentally correlated variation in levels of enzyme activity, suggest a translational or post-translational mechanism of ODC regulation. Since no PEST sequence was detected in the Ci ODC, enzyme regulation by programmed protein degradation as reported for many other eukaryotic ODCs may not occur in this case. ODC activity appears to play a key role in the morphogenesis of Ci, and the enzyme could be a rational target for therapy of disseminated coccidioidomycosis.
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PMID:Sequence, expression and functional analysis of the Coccidioides immitis ODC (ornithine decarboxylase) gene. 1072 38

Control of enzymatic function by peptide hormones can occur at a number of different levels and can involve diverse pathways that regulate cleavage, intracellular trafficking, and protein degradation. Gastrin is a peptide hormone that binds to the cholecystokinin B-gastrin receptor and regulates the activity of L-histidine decarboxylase (HDC), the enzyme that produces histamine. Here we show that gastrin can increase the steady-state levels of at least six HDC isoforms without affecting HDC mRNA levels. Pulse-chase experiments indicated that HDC isoforms are rapidly degraded and that gastrin-dependent increases are due to enhanced isoform stability. Deletion analysis identified two PEST domains (PEST1 and PEST2) and an intracellular targeting domain (ER2) which regulate HDC protein expression levels. Experiments with PEST domain fusion proteins demonstrated that PEST1 and PEST2 are strong and portable degradation-promoting elements which are positively regulated by both gastrin stimulation and proteasome inhibition. A chimeric protein containing the PEST domain of ornithine decarboxylase was similarly affected, indicating that gastrin can regulate the stability of other PEST domain-containing proteins and does so independently of antizyme/antizyme inhibitor regulation. At the same time, endoplasmic reticulum localization of a fluorescent chimera containing the ER2 domain of HDC was unaltered by gastrin stimulation. We conclude that gastrin stabilization of HDC isoforms is dependent upon two transferable and sequentially unrelated PEST domains that regulate degradation. These experiments revealed a novel regulatory mechanism by which a peptide hormone such as gastrin can disrupt the degradation function of multiple PEST-domain-containing proteins.
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PMID:Amino- and carboxy-terminal PEST domains mediate gastrin stabilization of rat L-histidine decarboxylase isoforms. 1084 18

The cDNA encoding ornithine decarboxylase (ODC; EC 4.1.1.17), a key enzyme in putrescine and polyamine biosynthesis, has been cloned from Nicotiana glutinosa (GenBank AF 323910), and was expressed in Escherichia coli. The amino acid sequence of N. glutinosa ODC showed 90% identity with Datura stramonium ODC, and 44% identity with human ODC. N. glutinosa ODC did not possess the PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] found in mammalian ODCs, which are thought to be involved in rapid degradation of the protein. The purified ODC was a homodimeric protein, having a native M(r) of 92000. Kinetic studies of ODC showed that N. glutinosa ODC decarboxylated both l-ornithine and l-lysine with K(m) values of 562 microM and 1592 microM at different optimal pH values of 8.0 and 6.8 respectively. ODC activity was completely and irreversibly inhibited by alpha-difluoromethylornithine (K(i) 1.15 microM), showing a competitive inhibition pattern. Site-directed mutagenesis was performed on ODC to introduce mutations at conserved lysine (Lys(95)) and cysteine (Cys(96), Cys(338) and Cys(377)) residues, chosen by examination of the conserved sequence, which were proven by chemical modification to be involved in enzymic activity. Except for Cys(96), each mutation caused a substantial loss in enzyme activity. Most notably, Lys(95) increased the K(m) for l-ornithine by 16-fold and for l-lysine by 3-fold, with 100-fold and 2.8-fold decreases in the k(cat) for ODC and lysine decarboxylase (LDC) activity respectively. The Cys(377)-->Ala mutant possessed a k(cat) that was lowered by 23-fold, and the K(m) value was decreased by 1.4-fold for l-ornithine. The three-dimensional model of ODC protein constructed on the basis of the crystal structure of Trypanosoma brucei, mouse and human ODCs localized the four residues in the active-site cleft. This is the first work carried out on active-site residues of plant ODC, where ODC and LDC activities occur in the same catalytic site.
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PMID:Identification of essential active-site residues in ornithine decarboxylase of Nicotiana glutinosa decarboxylating both L-ornithine and L-lysine. 1173 57

Polyamines are required for cellular growth and differentiation. In mammals and fungi they are synthesized via a pathway involving ornithine decarboxylase (ODC), which transforms ornithine into putrescine. We have cloned and disrupted the gene coding for ODC in Yarrowia lipolytica to analyze the role of polyamines in dimorphism of this fungus. Substrate- and cofactor-binding motifs, as well as two putative PEST boxes were identified in the amino acid sequence. A single transcript 1.7 kb in size was identified by Northern hybridization, and confirmed by rapid amplification of cDNA ends (RACE). Null mutants lacked ODC activity and behaved as polyamine auxotrophs. When low levels of polyamines were supplied to the null mutant, only yeast-like, but not mycelial growth was sustained. This phenomenon was confirmed by introduction of the YlODC gene under the control of an inducible promoter into the null mutant.
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PMID:Disruption of gene YlODC reveals absolute requirement of polyamines for mycelial development in Yarrowia lipolytica. 1270 44

The ectopic expression of antibody fragments inside mammalian cells (intrabodies) is a challenging approach for probing and modulating target activities. We previously described the shuttling activity of intracellularly expressed Escherichia coli beta-galactosidase conferred by the single-chain Fv (scFv) fragment 13R4 equipped with nuclear import/export signals. Here, by appending to scFvs the proteolytic PEST signal sequence (a protein region rich in proline, glutamic acid, serine and threonine) of mouse ornithine decarboxylase, we tested whether short-lived or destabilized intrabodies could affect the steady-state level of target by redirecting it to the proteasomes. In the absence of antigen, the half-life of the modified scFv 13R4, relative to untagged molecules, was considerably reduced in vivo. However, after coexpression with either cytoplasmic or nuclear antigen, the destabilized 13R4 fragments were readily maintained in the cell and strictly colocalized with beta-galactosidase. Analysis of destabilized site-directed mutants, that were as soluble as 13R4 in the intracellular context, demonstrated that binding to antigen was essential for survival under these conditions. This unique property allowed specific detection of beta-galactosidase, even when expressed at low level in stably transformed cells, and permitted isolation by flow cytometry from a transfected cell mixture of those living cells specifically labeled with bound intrabody. Altogether, we show that PEST-tagged intrabodies of sufficient affinity and solubility are powerful tools for imaging the presence and likely the dynamics of protein antigens that are resistant to proteasomal degradation in animal cells.
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PMID:Extended half-life upon binding of destabilized intrabodies allows specific detection of antigen in mammalian cells. 1594 19

We have developed a systematic and generic way to improve recombinant protein productivities in stable transfections by applying mRNA and protein destabilizing elements to reduce selection marker expression strength. Interferon-gamma (IFNgamma) expression vectors containing different combinations of AU-rich elements (ARE) and mouse ornithine decarboxylase (MODC) PEST region on the amplifiable dihydrofolate reductase (dhfr) selection marker were stably transfected into CHO-DG44 cells. Improvements in specific IFNgamma productivities were 1.7-, 6.6- and 13.3-fold with the application of ARE, MODC PEST, and both ARE and MODC PEST, respectively. To further enhance productivities, compatibility of the destabilizing sequences with methotrexate (MTX) amplification was validated by amplifying the transfected cells to 50nM MTX. A 14- to 27-fold increase in specific IFNgamma productivities were observed after amplification, indicating the compatibility of the two systems. A high specific IFNgamma productivity of 1.05pg/cell/day was also attained by the amplified cell pool with both ARE and MODC PEST.
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PMID:Application of destabilizing sequences on selection marker for improved recombinant protein productivity in CHO-DG44. 1736 64

Targeting of a DNA vaccine encoded protein for degradation via the proteasome is attempted since it may enhance the immunogenicity of the vaccine. We have fused HIV-1 reverse transcriptase (RT) to mouse ornithine decarboxylase (ODC), a protein rapidly degraded by proteasome in an ubiquitine-independent fashion, to enhance the introduction of RT into the MHC class I pathway. We also designed a fusion of RT with two short signals from the C-terminus of ODC (ODCsig) representing a minimal proteasome-targeting moiety of ODC (PEST signal). Fusion to ODC or ODC signal domain led to a marked enhancement of RT degradation. Plasmids encoding RT-ODC and RT-ODCsig chimera were used to immunize BALB/c mice. The administration of the plasmids was not associated with autoimmune disease. Moreover, mice receiving RT-ODCsig gene mounted a mixed Th1/Th2 response characterized by the in vitro secretion of IFN-gamma, IL-2, TNF-alpha, IL-4, and IL-10 upon stimulation of splenocytes with RT protein or RT derived peptides. Serum titers of 10(2) to 10(3) were observed in more than 50% of animals in that group, whereas fewer animals mounted an anti-RT response in the RT-ODC gene immunized group. Chimeras of the type described here can, therefore, be used in vaccinations aiming to induce HIV-1 RT-specific immune response.
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PMID:HIV-1 reverse transcriptase artificially targeted for proteasomal degradation induces a mixed Th1/Th2-type immune response. 1846 38

A method combining the use of a destabilized dihydrofolate reductase (DHFR) selection marker with methotrexate (MTX) amplification to generate high-expressing cells is described here. The selection marker expression is weakened with the use of the murine ornithine decarboxylase PEST region and AU-rich element to target the DHFR protein and mRNA, respectively, for degradation in the cell. Cells that produce higher levels of DHFR protein, and the adjoining recombinant protein gene, can compensate for the more rapid turnover of the DHFR protein and survive the selection process. This effect can complement MTX amplification to reduce the amount of MTX and shorten the time needed to generate a high-expressing clone. The gene of interest is first inserted into an expression vector that contains a destabilized DHFR selection marker. The resulting expression vector is then linearized and transfected into suspension CHO-DG44 cells. Selection is performed by culturing the cells in a selection medium lacking hypoxanthine and thymidine. Low concentrations of MTX are then used to amplify the transfected genes for increased protein expression. A single cell cloning protocol is also described. This can be used after each stage of MTX amplification to isolate high-expressing clones that are also consistent producers over longer culture periods.
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PMID:Generation of high-expressing cells by methotrexate amplification of destabilized dihydrofolate reductase selection marker. 2198 53


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